Compositions, methods, and systems for bead formation using improved polymers

ABSTRACT

The present disclosure provides systems and methods for making a hydrogel comprising a cell, cell nucleus, or one or more components derived from a cell or cell nucleus. A method for making a hydrogel may comprise providing a cell or cell nucleus, a first polymer, wherein the first polymer comprises a plurality of first crosslink precursors, each of the plurality of first crosslink precursors comprising an azide group; providing a second polymer, wherein the second polymer comprises a plurality of second crosslink precursors, each of the plurality of second crosslink precursors comprising an alkyne group; and crosslinking the first polymer and the second polymer via a reaction between a first section of the first crosslink precursors and a second section of the second crosslink precursors, thereby providing the hydrogel comprising the cell or cell nucleus.

CROSS-REFERENCE

This application is a continuation-in-part of U.S. patent applicationSer. No. 16/178,430, filed Nov. 1, 2018, which claims priority as acontinuation of International Patent Application PCT/US2018/054458,filed Oct. 4, 2018, which claims priority to U.S. Provisional PatentApplication No. 62/687,161, filed Jun. 19, 2018, and U.S. ProvisionalPatent Application No. 62/568,021, filed Oct. 4, 2017, each of which isentirely incorporated herein by reference for all purposes.

BACKGROUND

Samples may be processed for various purposes, such as identification ofa type of sample of moiety within the sample. The sample may be abiological sample. The biological samples may be processed for variouspurposes, such as detection of a disease (e.g., cancer) oridentification of a particular species. There are various approaches forprocessing samples, such as polymerase chain reaction (PCR) andsequencing.

Biological samples may be processed within various reactionenvironments, such as partitions. Partitions may be wells or droplets.Droplets or wells may be employed to process biological samples in amanner that enables the biological samples to be partitioned andprocessed separately. For example, such droplets may be fluidicallyisolated from other droplets, enabling accurate control of respectiveenvironments in the droplets.

Biological samples in partitions may be subjected to various processes,such as chemical processes or physical processes. Samples in partitionsmay be subjected to heating or cooling, or chemical reactions, such asto yield species that may be qualitatively or quantitatively processed.

SUMMARY

A hydrogel matrix (including a bead) can create a semi-open systemcapable of enclosing a large molecule within the boundary of the matrix,while allowing a small molecule to permeate the matrix. The largemolecule can be a biological sample, including, for example, a cell, alarge protein or a long nucleic acid. The small molecule can be, suchas, for example, a reagent, a smaller protein, or a shorter nucleicacid. An enzyme, for example, may be small enough to permeate thematrix. The hydrogel matrix can also comprise a labile bond such thatafter the hydrogel matrix is degraded, the enclosed large molecule canbe released from the confine of the matrix into the surroundingenvironment. Provided herein are methods, systems and compositions forthe production of a hydrogel matrix capable of enclosing a largemolecule and allowing a small molecule to permeate the matrix.

In some aspects, the present disclosure provides a gel, comprising: (a)a cell, a cell nucleus, or one or more constituents derived from a cell;(b) two or more polymers; and (c) a plurality of linkers, each of saidplurality of linkers comprising a 1,2,3-triazole moiety, wherein saidlinkers crosslink said two or more polymers. In some embodiments, eachof said two or more polymers independently comprises at least oneselected from the group consisting of a polyolefin, an olefin copolymer,an acrylic, a vinyl polymer, a polyester, a polycarbonate, a polyamide,a polyimide, a formaldehyde resin, a polyurethane, an ether polymer, acellulosic, a thermoplastic elastomer, and a thermoplastic polyurethane.In some embodiments, each of said two or more polymers is,independently, a polyacrylamide. In some embodiments, each of saidplurality of linkers is independently connected to an amide of said twoor more polymers. In some embodiments, each of said plurality of linkerscomprise a labile bond. In some embodiments, said labile bond is achemically labile bond, a thermally labile bond, or a photo-labile bond.In some embodiments, said labile bond comprises a disulfide bond. Insome embodiments, said 1,2,3-triazole moiety is formed by a process oftreating an azide group with an alkyne group in conditions sufficientfor forming said 1,2,3-triazole moiety. In some embodiments, the gelfurther comprises at least one reagent enclosed within said gel.

In some embodiments, said gel is a hydrogel. In some embodiments, saidgel further comprises a charged species. In some embodiments, saidcharged species is positively charged. In some embodiments, said chargedspecies comprises trimethylammonium. In some embodiments, said chargedspecies is negatively charged. In some embodiments, said charged speciescomprises phosphate. In some embodiments, said charged species isattached to said polymer or gel network. In some embodiments, at leastone of said two or more polymers is an electrically charged polymer. Insome embodiments, said electrically charged polymer is a positivelycharged polymer. In some embodiments, said positively charged polymercomprises chitosan or polyethyleneimine In some embodiments, saidelectrically charged polymer is a negatively charged polymer. In someembodiments, said a negatively charged polymer comprises alginate. Insome embodiments, at least one of said two or more polymers comprises anelectrically charged moiety, and wherein said electrically chargedmoiety is connected to said at least one of said two or more polymers bya linker. In some embodiments, said linker comprises a labile bondcapable of cleaving said electrically charged moiety from said at leastone of said two or more polymers. In some embodiments, said labile bondis a chemically labile bond, a thermally labile bond, or a photo-labilebond.

In some aspects, the present disclosure provides a method of forming agel comprising a cell or a cell nucleus, comprising: (a) providing (i) afirst polymer, wherein said first polymer comprises a plurality of firstcrosslink precursors, each of said plurality of first crosslinkprecursors comprising an azide group; (ii) a second polymer, whereinsaid second polymer comprises a plurality of second crosslinkprecursors, each of said plurality of second crosslink precursorscomprising an alkyne group; and (iii) said cell or said cell nucleus;(b) crosslinking said first polymer and said second polymer via areaction between a first section of said first crosslink precursors anda second section of said second crosslink precursors, thereby formingsaid gel comprising said cell or said cell nucleus. In some embodiments,said first polymer and said second polymer independently comprise atleast one selected from the group consisting of a polyolefin, an olefincopolymer, an acrylic, a vinyl polymer, a polyester, a polycarbonate, apolyamide, a polyimide, a formaldehyde resin, a polyurethane, an etherpolymer, a cellulosic, a thermoplastic elastomer, and a thermoplasticpolyurethane. In some embodiments, said first polymer or said secondpolymer further comprise a labile bond. In some embodiments, said firstpolymer and said second polymer further comprise a labile bond. In someembodiments, said labile bond is a disulfide bond. In some embodiments,at least about 80% of said labile bond remains intact during saidreaction in (b). In some embodiments, said reaction forms a1,2,3-triazole between said azide and said alkyne. In some embodiments,the method further comprises prior to (b), providing a catalystconfigured to catalyze said reaction in (b). In some embodiments, themethod further comprises subsequent to (b), removing said catalystand/or a derivative thereof from said gel. In some embodiments, said gelis formed from a plurality of said first polymers and a plurality ofsaid second polymers. In some embodiments, said gel is a hydrogel.

In some embodiments, the method further comprises subsequent to (b),lysing said cell or said cell nucleus to release one or more cell orcell nucleus constituents into said gel. In some embodiments, said oneor more cell or cell nucleus constituents comprises a nucleic acid. Insome embodiments, said nucleic acid comprises a ribonucleic acid. Insome embodiments, said ribonucleic acid is a messenger ribonucleic acid(mRNA). In some embodiments, said ribonucleic acid is a microribonucleic acid (miRNA). In some embodiments, said nucleic acidcomprises a deoxyribonucleic acid (DNA). In some embodiments, said DNAis genomic DNA. In some embodiments, said one or more cell or cellnucleus constituents comprises chromatin. In some embodiments, said oneor more cell or cell nucleus constituents comprises a protein. In someembodiments, said one or more cell or cell nucleus constituents iscapable of being retained within said gel. In some embodiments, said oneor more cell or cell nucleus constituents is capable of being retainedwithin said gel for at least 1, at least 2, at least 3, at least 4, atleast 5, at least 12, or at least 24 hours. In some embodiments, themethod further comprises denaturing said DNA. In some embodiments, saiddenaturing comprises contacting said gel with a chemical reagent. Insome embodiments, said chemical reagent is an alkaline reagent. In someembodiments, the method further comprises subsequent to (b),permeabilizing said cell or said cell nucleus. In some embodiments, themethod further comprises prior to (b), co-partitioning said firstpolymer, said second polymer, and said cell or cell nucleus into apartition. In some embodiments, said partition is a well. In someembodiments, said partition is an aqueous droplet in an emulsion. Insome embodiments, said partition comprises a reagent configured tocatalyze said reaction in (b). In some embodiments, said emulsioncomprises an oil phase comprising a reagent comprising a copper (II)moiety, and wherein said aqueous droplet comprises a reducing agentcapable of reducing said copper (II) moiety to a copper (I) moiety,wherein said copper (I) moiety catalyzes said reaction in (b). In someembodiments, said emulsion comprises a reagent that facilitates thetransport of said copper (II) moiety from said oil phase into saidaqueous droplet. In some embodiments, the method further comprises priorto (b), lysing or permeabilizing said cell or cell nucleus in saidpartition.

In some aspects, the present disclosure provides a method for generatinga cell bead, comprising: (a) generating a partition comprising a cellfrom a plurality of cells or a nucleus from a plurality of nuclei, apolymeric or gel precursor, and a charged species; and (b) subjectingsaid partition to conditions sufficient to react said polymeric or gelprecursor to generate a polymer or gel network comprising (i) said cellor a derivative thereof, and (ii) said charged species, therebyproviding said cell bead. In some embodiments, said partition is among aplurality of partitions. In some embodiments, the method furthercomprises generating a plurality of cell beads from said plurality ofpartitions. In some embodiments, said charged species is positivelycharged. In some embodiments, said charged species comprisestrimethylammonium. In some embodiments, said charged species is(3-acrylamidopropyl) trimethylammonium. In some embodiments, saidcharged species is negatively charged. In some embodiments, said chargedspecies comprises phosphate. In some embodiments, said charged speciesis attached to said polymer or gel network. In some embodiments, saidcell bead comprises a plurality of chemical cross-linkers. In someembodiments, said charged species is attached to a chemical-cross linkerof said chemical cross-linkers. In some embodiments, the method furthercomprises prior to (b), subjecting said cell bead to conditionssufficient to lyse said cell or cell nucleus to release one or more cellor cell nucleus constituents into said cell bead.

In some embodiments, the method further comprises subsequent to (b),subjecting said cell bead to conditions sufficient to lyse said cell orcell nucleus to release one or more cell or cell nucleus constituentsinto said cell bead. In some embodiments, said one or more cell or cellnucleus constituents comprises a nucleic acid. In some embodiments, saidnucleic acid comprises ribonucleic acid. In some embodiments, saidribonucleic acid is a messenger ribonucleic acid. In some embodiments,said nucleic acid comprises a deoxyribonucleic acid. In someembodiments, said one or more cell or cell nucleus constituentscomprises a protein. In some embodiments, said one or more cell or cellnucleus constituents is capable of being retained within said cell bead.In some embodiments, said one or more cell or cell nucleus constituentsis capable of being retained within said cell bead for at least 1, atleast 2, at least 3, at least 4, at least 5, at least 12, or at least 24hours.

In some aspects, the present disclosure provides a method for generatinga cell bead, comprising: (a) generating a partition comprising (i) acell from a plurality of cells or a nucleus from a plurality of nucleiand (ii) an electrically charged polymeric or gel precursor; and (b)subjecting said partition to conditions sufficient to react saidpolymeric or gel precursor to generate an electrically charged polymeror gel network comprising said cell or said nucleus or a derivativethereof, thereby providing said cell bead. In some embodiments, saidelectrically charged polymeric or gel precursor comprises a positivecharge. In some embodiments, said electrically charged polymeric or gelprecursor comprises chitosan. In some embodiments, said electricallycharged polymeric or gel precursor comprises polyethyleneimine In someembodiments, said electrically charged polymeric or gel precursorcomprises a negative charge. In some embodiments, said electricallycharged polymeric or gel precursor comprises alginate. In someembodiments, the method further comprises prior to (b), subjecting saidcell bead to conditions sufficient to lyse said cell or cell nucleus torelease one or more cell or cell nucleus constituents into said cellbead. In some embodiments, the method further comprises subsequent to(b), subjecting said cell bead to conditions sufficient to lyse said orcell nucleus to release one or more cell or cell nucleus constituentsinto said cell bead. In some embodiments, said one or more cell or cellnucleus constituents a nucleic acid. In some embodiments, said nucleicacid comprises a ribonucleic acid. In some embodiments, said ribonucleicacid is a messenger ribonucleic acid. In some embodiments, said nucleicacid comprises a deoxyribonucleic acid. In some embodiments, said one ormore cell or cell nucleus constituents comprises a protein. In someembodiments, said one or more cell or cell nucleus constituents iscapable of being retained within said cell bead. In some embodiments,said one or more cell or cell nucleus constituents is capable of beingretained within said cell bead for at least 1, at least 2, at least 3,at least 4, at least 5, at least 12, or at least 24 hours.

In some aspects, the present disclosure provides a composition for usein analyzing one or more components from a cell, comprising a cell beadcomprising a polymerized or cross-linked polymer network comprising acell, a cell nucleus, or one or more constituents derived from a cell ora cell nucleus, wherein said polymerized or cross-linked polymer networkis electrically charged. In some embodiments, said polymer network ispositively charged. In some embodiments, said polymer network comprisespolyethyleneimine In some embodiments, said polymer network compriseschitosan. In some embodiments, said polymer network is negativelycharged. In some embodiments, said polymer network comprises alginate.

In some aspects, the present disclosure provides a composition for usein analyzing one or more components from a cell, comprising a cell beadcomprising a polymerized or cross-linked polymer network comprising (i)a cell, a cell nucleus, or one or more constituents derived from a cellor a cell nucleus; and (ii) a charged species. In some embodiments, saidpolymer network comprises a chemical cross-linker. In some embodiments,said cell bead comprises a component from said cell attached to saidchemical cross-linker. In some embodiments, said charged species isattached to said polymer network. In some embodiments, said chargedspecies is covalently attached to said polymer network. In someembodiments, said charges species is attached to a component from saidcell. In some embodiments, said charged species is non-covalentlyattached to said component from said cell. In some embodiments, saidcharged species is positively charged. In some embodiments, said chargedspecies comprises trimethylammonium. In some embodiments, said chargedspecies is (2-Aminoethyl)trimethylammonium. In some embodiments, saidcharged species is (3-Acrylamidopropyl)trimethylammonium. In someembodiments, said charged species is negatively charged. In someembodiments, said charged species comprises phosphate.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

Incorporation by Reference

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “Figure” and “FIG.” herein), of which:

FIG. 1 shows an example of a microfluidic channel structure forpartitioning individual biological particles.

FIG. 2 shows an example of a microfluidic channel structure fordelivering barcode carrying beads to droplets.

FIG. 3 shows an example of a microfluidic channel structure forco-partitioning biological particles and reagents.

FIG. 4 shows an example of a microfluidic channel structure for thecontrolled partitioning of beads into discrete droplets.

FIG. 5 shows an example of a microfluidic channel structure forincreased droplet generation throughput.

FIG. 6 shows another example of a microfluidic channel structure forincreased droplet generation throughput.

FIG. 7 shows an example of a microfluidic channel structure forco-partitioning biological particles and reagents to form dropletsconfigured to form hydrogels.

FIG. 8 shows an example hydrogel composition and steps for forminghydrogels in a droplet.

FIG. 9 shows an example method for generating a click chemistry polymernetwork.

FIG. 10 illustrates an example method for generating a cell bead of thepresent disclosure.

FIGS. 11A-B show an example charged hydrogel polymer network. FIG. 11Ashows a schematic of a cell bead comprising positively charged speciesattached to a polymer network. FIG. 11B shows a schematic of a cell beadcomprising negatively charged species attached to a polymer network.

FIGS. 12A-B show an additional example charged hydrogel polymer network.FIG. 12A shows a schematic of a cell bead comprising positively chargesspecies attached to disulfide containing chemical cross-linkers. FIG.12B shows a schematic of a cell bead comprising negatively chargesspecies attached to disulfide containing chemical cross-linkers.

FIG. 13 illustrates an example process for generating dropletscomprising constituents from a cell.

FIG. 14 illustrates another example process for generating dropletscomprising constituents from a cell.

FIG. 15 illustrates an example process for generating cell beadscomprising complementary deoxyribonucleic acid.

FIG. 16A schematically illustrates an example method for generatingdroplets comprising a barcoded bead and a cell bead. FIG. 16Billustrates an example microfluidic architecture for generating cellbeads. FIG. 16C illustrates an example microfluidic architecture forgenerating droplets comprising barcoded beads and cell beads. FIG. 16Dillustrates an example droplet generation process generating dropletscomprising a barcoded bead and a cell bead using the architecture shownin FIG. 16C.

FIG. 17 shows an example process for generating a cell bead and apartition comprising a cell bead and a gel bead.

FIG. 18 shows an example computer system that is programmed or otherwiseconfigured to implement methods and systems provided herein.

FIG. 19 shows a representative microscope image of a cell bead enclosinga cell.

FIG. 20 shows experimental sequencing results from DNA obtained from acell comprised in a cell bead.

FIG. 21A diagrams an example workflow for generating cell beads. FIG.21B shows imaging results from cell beads generated without cellcentering. FIG. 21C shows imaging results from cell beads generated withcell centering.

FIG. 22 shows a graph of results from the cell centering experiments ofExample 4.

FIG. 23 shows a graph of results from the cell bead generationexperiments of Example 5.

FIG. 24 shows a graph of results from the cell bead generationexperiments of Example 6.

FIG. 25A shows the results from a cell bead generation experimentdescribed in Example 7 comprising the use of varying sodium ascorbateconcentrations. FIG. 25B shows the results from a cell bead generationexperiment described in Example 7 comprising the use of varying gelationtimes. FIG. 25C shows the results from a cell bead generation experimentdescribed in Example 7 comprising the use of varying THPTAconcentrations. FIG. 25D shows the results from a cell bead generationexperiment described in Example 7 comprising the use of varying sodiumascorbate concentrations.

FIG. 26A shows the results from a cell bead generation experimentdescribed in Example 7 comprising the use of varying CuAcAcconcentrations. FIG. 26B shows the results from a cell bead generationexperiment described in Example 7 comprising the use of varying gelationtimes.

FIG. 27A shows the results from a cell bead generation experimentdescribed in Example 8 comprising the use of varying THPTAconcentrations. FIG. 27B shows the results from a cell bead generationexperiment described in Example 8 comprising the use of varying sodiumascorbate concentrations. FIG. 27C shows the results from a cell beadgeneration experiment described in Example 8 comprising the use ofvarying sodium ascorbate concentrations. FIG. 27D shows the results froma cell bead generation experiment described in Example 8 comprising theuse of varying sodium ascorbate concentrations.

FIG. 28A shows the results from a cell bead generation experimentdescribed in Example 8 comprising the use of varying THPTAconcentrations. FIG. 28B shows the results from a cell bead generationexperiment described in Example 8 comprising the use of varying gelationtimes.

FIG. 29 shows an exemplary low-copper concentration click chemistrycrosslinking reaction using an azide-picolyl modified linker.

FIG. 30 shows an exemplary copper-free click chemistry crosslinkingreaction using azide-modified and DBCO-modified linkers.

FIG. 31A shows results of low-copper concentration click chemistrycrosslinking using azide-picolyl modified linkers as described inExample 11.

FIG. 31B shows results of low-copper concentration click chemistrycrosslinking using azide-picolyl modified linkers as described inExample 11.

FIG. 32 shows an exemplary click chemistry crosslinking reaction to formcrosslinks with a labile carbamate bond, and subsequent cleavage of thecarbamate with DETA and heat.

FIG. 33 shows results of degradation of carbamate crosslinks with DETAand heat as described in Example 12.

FIG. 34A shows an exemplary propargyl-polypeptide linker that can form aprotease cleavable crosslink via click chemistry.

FIG. 34B shows formation of an exemplary protease cleavable polypeptidecrosslink via click chemistry and subsequent selective proteasecatalyzed degradation of the crosslink.

FIG. 35 shows an exemplary reaction forming via click chemistrycrosslinked polymers with one of the polymers modified with a poly-Toligonucleotide capture reagent attached via click chemistry.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

Where values are described as ranges, it will be understood that suchdisclosure includes the disclosure of all possible sub-ranges withinsuch ranges, as well as specific numerical values that fall within suchranges irrespective of whether a specific numerical value or specificsub-range is expressly stated.

The term “barcode,” as used herein, generally refers to a label, oridentifier, that conveys or is capable of conveying information about ananalyte. A barcode can be part of an analyte. A barcode can beindependent of an analyte. A barcode can be a tag attached to an analyte(e.g., nucleic acid molecule) or a combination of the tag in addition toan endogenous characteristic of the analyte (e.g., size of the analyteor end sequence(s)). A barcode may be unique. Barcodes can have avariety of different formats. For example, barcodes can include:polynucleotide barcodes; random nucleic acid and/or amino acidsequences; and synthetic nucleic acid and/or amino acid sequences. Abarcode can be attached to an analyte in a reversible or irreversiblemanner. A barcode can be added to, for example, a fragment of adeoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before,during, and/or after sequencing of the sample. Barcodes can allow foridentification and/or quantification of individual sequencing-reads inreal time.

The term “real time,” as used herein, can refer to a response time ofless than about 1 second, a tenth of a second, a hundredth of a second,a millisecond, or less. The response time may be greater than 1 second.In some instances, real time can refer to simultaneous or substantiallysimultaneous processing, detection or identification.

The term “subject,” as used herein, generally refers to an animal, suchas a mammal (e.g., human) or avian (e.g., bird), or other organism, suchas a plant. The subject can be a vertebrate, a mammal, a rodent (e.g., amouse), a primate, a simian or a human. Animals may include, but are notlimited to, farm animals, sport animals, and pets. A subject can be ahealthy or asymptomatic individual, an individual that has or issuspected of having a disease (e.g., cancer) or a pre-disposition to thedisease, and/or an individual that is in need of therapy or suspected ofneeding therapy. A subject can be a patient.

The term “genome,” as used herein, generally refers to genomicinformation from a subject, which may be, for example, at least aportion or an entirety of a subject's hereditary information. A genomecan be encoded either in DNA or in RNA. A genome can comprise codingregions (e.g., that code for proteins) as well as non-coding regions. Agenome can include the sequence of all chromosomes together in anorganism. For example, the human genome ordinarily has a total of 46chromosomes. The sequence of all of these together may constitute ahuman genome.

The terms “adaptor(s)”, “adapter(s)” and “tag(s)” may be usedsynonymously. An adaptor or tag can be coupled to a polynucleotidesequence to be “tagged” by any approach, including ligation,hybridization, or other approaches.

The term “sequencing,” as used herein, generally refers to methods andtechnologies for determining the sequence of nucleotide bases in one ormore polynucleotides. The polynucleotides can be, for example, nucleicacid molecules such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), including variants or derivatives thereof (e.g., single strandedDNA). Sequencing can be performed by various systems currentlyavailable, such as, without limitation, a sequencing system byIllumina®, Pacific Biosciences (PACBIO®), OXFORD NANOPORE®, or LifeTechnologies (ION TORRENT®). Alternatively, or in addition, sequencingmay be performed using nucleic acid amplification, polymerase chainreaction (PCR) (e.g., digital PCR, quantitative PCR, or real time PCR),or isothermal amplification. Such systems may provide a plurality of rawgenetic data corresponding to the genetic information of a subject(e.g., human), as generated by the systems from a sample provided by thesubject. In some examples, such systems provide sequencing reads (also“reads” herein). A read may include a string of nucleic acid basescorresponding to a sequence of a nucleic acid molecule that has beensequenced. In some situations, systems and methods provided herein maybe used with proteomic information.

The term “bead,” as used herein, generally refers to a particle. Thebead may be a solid or semi-solid particle. The bead may be a gel bead.The gel bead may include a polymer matrix (e.g., matrix formed bypolymerization or cross-linking). The bead may be formed of a polymericmaterial. The bead may be magnetic or non-magnetic. The bead may berigid. The bead may be flexible and/or compressible. The bead may bedisruptable or dissolvable.

The term “sample,” as used herein, generally refers to a biologicalsample of a subject. The biological sample may comprise any number ofmacromolecules, for example, cellular macromolecules. The biologicalsample may be a nucleic acid sample or protein sample. The biologicalsample may also be a carbohydrate sample or a lipid sample. Thebiological sample may be derived from another sample. The sample may bea tissue sample, such as a biopsy, core biopsy, needle aspirate, or fineneedle aspirate. The sample may be a fluid sample, such as a bloodsample, urine sample, or saliva sample. The sample may be a skin sample.The sample may be a cheek swab. The sample may be a plasma or serumsample. The sample may be a cell-free or cell free sample. A cell-freesample may include extracellular polynucleotides. Extracellularpolynucleotides may be isolated from a bodily sample that may beselected from the group consisting of blood, plasma, serum, urine,saliva, mucosal excretions, sputum, stool and tears.

The term “biological particle,” as used herein, generally refers to adiscrete biological system derived from a biological sample. Thebiological particle may be a virus. The biological particle may be acell or derivative of a cell. The biological particle may be anorganelle. The biological particle may be a rare cell from a populationof cells. The biological particle may be any type of cell, includingwithout limitation prokaryotic cells, eukaryotic cells, bacterial,fungal, plant, mammalian, or other animal cell type, mycoplasmas, normaltissue cells, tumor cells, or any other cell type, whether derived fromsingle cell or multicellular organisms. The biological particle may beor may include a matrix (e.g., a gel or polymer matrix) comprising acell or one or more constituents from a cell (e.g., cell bead), such asDNA, RNA, organelles, proteins, or any combination thereof, from thecell. The biological particle may be obtained from a tissue of asubject. The biological particle may be a hardened cell. Such hardenedcell may or may not include a cell wall or cell membrane. The biologicalparticle may include one or more constituents of a cell but may notinclude other constituents of the cell. An example of such constituentsis a nucleus or an organelle. A cell may be a live cell. The live cellmay be capable of being cultured, for example, being cultured whenenclosed in a gel or polymer matrix or cultured when comprising a gel orpolymer matrix.

The term “macromolecular constituent,” as used herein, generally refersto a macromolecule contained within a biological particle. Themacromolecular constituent may comprise a nucleic acid. Themacromolecular constituent may comprise DNA. The macromolecularconstituent may comprise RNA. The RNA may be messenger RNA (mRNA),ribosomal RNA (rRNA) or transfer RNA (tRNA). The RNA may be atranscript. The macromolecular constituent may comprise a protein. Themacromolecular constituent may comprise a peptide. The macromolecularconstituent may comprise a polypeptide.

The term “molecular tag,” as used herein, generally refers to a moleculecapable of binding to a macromolecular constituent. The molecular tagmay bind to the macromolecular constituent with high affinity. Themolecular tag may bind to the macromolecular constituent with highspecificity. The molecular tag may comprise a nucleotide sequence. Themolecular tag may comprise a nucleic acid sequence. The nucleic acidsequence may be at least a portion or an entirety of the molecular tag.The molecular tag may be a nucleic acid molecule or may be part of anucleic acid molecule. The molecular tag may be an oligonucleotide or apolypeptide. The molecular tag may comprise a DNA aptamer. The moleculartag may be or comprise a primer. The molecular tag may be, or comprise,a protein. The molecular tag may comprise a polypeptide. The moleculartag may be a barcode.

Provided herein are compositions and methods for forming hydrogelmatrices (including beads) by emulsion gelation. In addition, thehydrogel matrices can enclose a large molecule such as a biologicalsample. The biological sample can be a cell, a large protein, or a longnucleic acid. The hydrogel matrices can comprise a cell or one or morecomponents from a cell (e.g., a cell bead). The hydrogel matrices canallow a smaller molecule to permeate the matrices. The smaller moleculecan be a reagent, a smaller protein, or a shorter nucleic acid. Thesmaller protein can be an enzyme. The hydrogel can be degradable.

In an aspect, the present disclosure provides a composition of adegradable hydrogel comprising two or more polymers, and a plurality oflinkers configured to form crosslinks. Each of the plurality of linkerscan comprise a labile bond and a 1,2,3-triazole moiety. The two or morepolymers can be crosslinked by such linkers.

In an aspect, the present disclosure provides a method of forming ahydrogel. The method can comprise (a) providing a first polymer, whereinthe first polymer comprises a plurality of first crosslink precursors;(b) providing a second polymer, wherein the second polymer comprises aplurality of second crosslink precursors; and (c) crosslinking the firstpolymer and the second polymer via a reaction between a first section ofthe first crosslink precursors and a second section of the secondcrosslink precursors, thereby forming the hydrogel.

In an aspect, the systems and methods described herein provide for thecompartmentalization, depositing, or partitioning of macromolecularconstituent contents of individual biological particles into discretecompartments or partitions (referred to interchangeably herein aspartitions), where each partition maintains separation of its owncontents from the contents of other partitions. The partition can be adroplet in an emulsion. A partition may comprise one or more otherpartitions.

A partition of the present disclosure may comprise biological particlesand/or macromolecular constituents thereof. A partition may comprise oneor more gel beads. A partition may comprise one or more cell beads. Apartition may include a single gel bead, a single cell bead, or both asingle cell bead and single gel bead. A cell bead can be a biologicalparticle and/or one or more of its macromolecular constituents encasedinside of a gel or polymer matrix, such as via polymerization of adroplet containing the biological particle and precursors capable ofbeing polymerized or gelled. Unique identifiers, such as barcodes, maybe injected into the droplets previous to, subsequent to, orconcurrently with droplet generation, such as via a microcapsule (e.g.,bead), as described further below. Microfluidic channel networks (e.g.,on a chip) can be utilized to generate partitions as described herein.Alternative mechanisms may also be employed in the partitioning ofindividual biological particles, including porous membranes throughwhich aqueous mixtures of cells are extruded into non-aqueous fluids.

The partitions can be flowable within fluid streams. The partitions maycomprise, for example, micro-vesicles that have an outer barriersurrounding an inner fluid center or core. In some cases, the partitionsmay comprise a porous matrix that is capable of entraining and/orretaining materials within its matrix. The partitions can comprisedroplets of aqueous fluid within a non-aqueous continuous phase (e.g.,oil phase). The partitions can comprise droplets of a first phase withina second phase, wherein the first and second phases are immiscible. Avariety of different vessels are described in, for example, U.S. PatentApplication Publication No. 2014/0155295, which is entirely incorporatedherein by reference for all purposes. Emulsion systems for creatingstable droplets in non-aqueous or oil continuous phases are describedin, for example, U.S. Patent Application Publication No. 2010/0105112,which is entirely incorporated herein by reference for all purposes.

In the case of droplets in an emulsion, allocating individual biologicalparticles to discrete partitions may in one non-limiting example beaccomplished by introducing a flowing stream of biological particles inan aqueous fluid into a flowing stream of a non-aqueous fluid, such thatdroplets are generated at the junction of the two streams. By providingthe aqueous stream at a certain concentration and/or flow rate ofbiological particles, the occupancy of the resulting partitions (e.g.,number of biological particles per partition) can be controlled. Wheresingle biological particle partitions are used, the relative flow ratesof the immiscible fluids can be selected such that, on average, thepartitions may contain less than one biological particle per partitionin order to ensure that those partitions that are occupied are primarilysingly occupied. In some cases, partitions among a plurality ofpartitions may contain at most one biological particle (e.g., bead, cellor cellular material). In some embodiments, the relative flow rates ofthe fluids can be selected such that a majority of partitions areoccupied, for example, allowing for only a small percentage ofunoccupied partitions. The flows and channel architectures can becontrolled as to ensure a given number of singly occupied partitions,less than a certain level of unoccupied partitions and/or less than acertain level of multiply occupied partitions.

FIG. 1 shows an example of a microfluidic channel structure 100 forpartitioning individual biological particles. The channel structure 100can include channel segments 102, 104, 106 and 108 communicating at achannel junction 110. In operation, a first aqueous fluid 112 thatincludes suspended biological particles (or cells) 114 may betransported along channel segment 102 into junction 110, while a secondfluid 116 that is immiscible with the aqueous fluid 112 is delivered tothe junction 110 from each of channel segments 104 and 106 to creatediscrete droplets 118, 120 of the first aqueous fluid 112 flowing intochannel segment 108, and flowing away from junction 110. The channelsegment 108 may be fluidically coupled to an outlet reservoir where thediscrete droplets can be stored and/or harvested. A discrete dropletgenerated may include an individual biological particle 114 (such asdroplets 118). A discrete droplet generated may include more than oneindividual biological particle 114 (not shown in FIG. 1). A discretedroplet may contain no biological particle 114 (such as droplet 120).Each discrete partition may maintain separation of its own contents(e.g., individual biological particle 114) from the contents of otherpartitions.

The second fluid 116 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets118, 120. Examples of particularly useful partitioning fluids andfluorosurfactants are described, for example, in U.S. Patent ApplicationPublication No. 2010/0105112, which is entirely incorporated herein byreference for all purposes.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 100 may have other geometries. For example, amicrofluidic channel structure can have more than one channel junction.For example, a microfluidic channel structure can have 2, 3, 4, or 5channel segments each carrying biological particles, cell beads, and/orgel beads that meet at a channel junction. Fluid may be directed flowalong one or more channels or reservoirs via one or more fluid flowunits. A fluid flow unit can comprise compressors (e.g., providingpositive pressure), pumps (e.g., providing negative pressure),actuators, and the like to control flow of the fluid. Fluid may also orotherwise be controlled via applied pressure differentials, centrifugalforce, electrokinetic pumping, vacuum, capillary or gravity flow, or thelike.

The generated droplets may comprise two subsets of droplets: (1)occupied droplets 118, containing one or more biological particles 114,and (2) unoccupied droplets 120, not containing any biological particles114. Occupied droplets 118 may comprise singly occupied droplets (havingone biological particle) and multiply occupied droplets (having morethan one biological particle). As described elsewhere herein, in somecases, the majority of occupied partitions can include no more than onebiological particle per occupied partition and some of the generatedpartitions can be unoccupied (of any biological particle). In somecases, though, some of the occupied partitions may include more than onebiological particle. In some cases, the partitioning process may becontrolled such that fewer than about 25% of the occupied partitionscontain more than one biological particle, and in many cases, fewer thanabout 20% of the occupied partitions have more than one biologicalparticle, while in some cases, fewer than about 10% or even fewer thanabout 5% of the occupied partitions include more than one biologicalparticle per partition.

In some cases, it may be desirable to minimize the creation of excessivenumbers of empty partitions, such as to reduce costs and/or increaseefficiency. While this minimization may be achieved by providing asufficient number of biological particles (e.g., biological particles114) at the partitioning junction 110, such as to ensure that at leastone biological particle is encapsulated in a partition, the Poissoniandistribution may expectedly increase the number of partitions thatinclude multiple biological particles. As such, where singly occupiedpartitions are to be obtained, at most about 95%, 90%, 85%, 80%, 75%,70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% orless of the generated partitions can be unoccupied.

In some cases, the flow of one or more of the biological particles(e.g., in channel segment 102), or other fluids directed into thepartitioning junction (e.g., in channel segments 104, 106) can becontrolled such that, in many cases, no more than about 50% of thegenerated partitions, no more than about 25% of the generatedpartitions, or no more than about 10% of the generated partitions areunoccupied. These flows can be controlled so as to present anon-Poissonian distribution of single-occupied partitions whileproviding lower levels of unoccupied partitions. The above noted rangesof unoccupied partitions can be achieved while still providing any ofthe single occupancy rates described above. For example, in many cases,the use of the systems and methods described herein can create resultingpartitions that have multiple occupancy rates of less than about 25%,less than about 20%, less than about 15%, less than about 10%, and inmany cases, less than about 5%, while having unoccupied partitions ofless than about 50%, less than about 40%, less than about 30%, less thanabout 20%, less than about 10%, less than about 5%, or less.

As will be appreciated, the above-described occupancy rates are alsoapplicable to partitions that include both biological particles andadditional reagents, including, but not limited to, microcapsulescarrying barcoded nucleic acid molecules (e.g., oligonucleotides)(described in relation to FIG. 2). The occupied partitions (e.g., atleast about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% ofthe occupied partitions) can include both a microcapsule (e.g., bead)comprising barcoded nucleic acid molecules and a biological particle.

In another aspect, in addition to or as an alternative to droplet basedpartitioning, biological particles may be encapsulated within amicrocapsule that comprises an outer shell, layer or porous matrix inwhich is entrained one or more individual biological particles or smallgroups of biological particles. The microcapsule may include otherreagents. Encapsulation of biological particles may be performed by avariety of processes. Such processes combine an aqueous fluid containingthe biological particles with a polymeric precursor material that may becapable of being formed into a gel or other solid or semi-solid matrixupon application of a particular stimulus to the polymer precursor. Suchstimuli can include, for example, thermal stimuli (either heating orcooling), photo-stimuli (e.g., through photo-curing), chemical stimuli(e.g., through crosslinking, polymerization initiation of the precursor(e.g., through added initiators), or the like, and/or a combination ofthe above.

Preparation of microcapsules comprising biological particles may beperformed by a variety of methods. For example, air knife droplet oraerosol generators may be used to dispense droplets of precursor fluidsinto gelling solutions in order to form microcapsules that includeindividual biological particles or small groups of biological particles.Likewise, membrane based encapsulation systems may be used to generatemicrocapsules comprising encapsulated biological particles as describedherein. Microfluidic systems of the present disclosure, such as thatshown in FIG. 1, may be readily used in encapsulating cells as describedherein. In particular, and with reference to FIG. 1, the aqueous fluid112 comprising (i) the biological particles 114 and (ii) the polymerprecursor material (not shown) is flowed into channel junction 110,where it is partitioned into droplets 118, 120 through the flow ofnon-aqueous fluid 116. In the case of encapsulation methods, non-aqueousfluid 116 may also include an initiator (not shown) to causepolymerization and/or crosslinking of the polymer precursor to form themicrocapsule that includes the entrained biological particles. Examplesof polymer precursor/initiator pairs include those described in U.S.Patent Application Publication No. 2014/0378345, which is entirelyincorporated herein by reference for all purposes.

For example, in the case where the polymer precursor material comprisesa linear polymer material, such as a linear polyacrylamide, PEG, orother linear polymeric material, the activation agent may comprise across-linking agent, or a chemical that activates a cross-linking agentwithin the formed droplets. Likewise, for polymer precursors thatcomprise polymerizable monomers, the activation agent may comprise apolymerization initiator. For example, in certain cases, where thepolymer precursor comprises a mixture of acrylamide monomer with aN,N′-bis-(acryloyl)cystamine (BAC) comonomer, an agent such astetraethylmethylenediamine (TEMED) may be provided within the secondfluid streams 116 in channel segments 104 and 106, which can initiatethe copolymerization of the acrylamide and BAC into a cross-linkedpolymer network, or hydrogel.

Upon contact of the second fluid stream 116 with the first fluid stream112 at junction 110, during formation of droplets, the TEMED may diffusefrom the second fluid 116 into the aqueous fluid 112 comprising thelinear polyacrylamide, which will activate the crosslinking of thepolyacrylamide within the droplets 118, 120, resulting in the formationof gel (e.g., hydrogel) microcapsules, as solid or semi-solid beads orparticles entraining the cells 114. Although described in terms ofpolyacrylamide encapsulation, other ‘activatable’ encapsulationcompositions may also be employed in the context of the methods andcompositions described herein. For example, formation of alginatedroplets followed by exposure to divalent metal ions (e.g., Ca²⁺ ions),can be used as an encapsulation process using the described processes.Likewise, agarose droplets may also be transformed into capsules throughtemperature based gelling (e.g., upon cooling, etc.).

In some cases, encapsulated biological particles can be selectivelyreleasable from the microcapsule, such as through passage of time orupon application of a particular stimulus, that degrades themicrocapsule sufficiently to allow the biological particles (e.g.,cell), or its other contents to be released from the microcapsule, suchas into a partition (e.g., droplet). For example, in the case of thepolyacrylamide polymer described above, degradation of the microcapsulemay be accomplished through the introduction of an appropriate reducingagent, such as DTT or the like, to cleave disulfide bonds thatcross-link the polymer matrix. See, for example, U.S. Patent ApplicationPublication No. 2014/0378345, which is entirely incorporated herein byreference for all purposes.

The biological particle can be subjected to other conditions sufficientto polymerize or gel the precursors. The conditions sufficient topolymerize or gel the precursors may comprise exposure to heating,cooling, electromagnetic radiation, and/or light. The conditionssufficient to polymerize or gel the precursors may comprise anyconditions sufficient to polymerize or gel the precursors. Followingpolymerization or gelling, a polymer or gel may be formed around thebiological particle. The polymer or gel may be diffusively permeable tochemical or biochemical reagents. The polymer or gel may be diffusivelyimpermeable to macromolecular constituents of the biological particle.In this manner, the polymer or gel may act to allow the biologicalparticle to be subjected to chemical or biochemical operations whilespatially confining the macromolecular constituents to a region of thedroplet defined by the polymer or gel. The polymer or gel may includeone or more of disulfide cross-linked polyacrylamide, agarose, alginate,polyvinyl alcohol, polyethylene glycol (PEG)-diacrylate, PEG-acrylate,PEG-thiol, PEG-azide, PEG-alkyne, other acrylates, chitosan, hyaluronicacid, collagen, fibrin, gelatin, or elastin. The polymer or gel maycomprise any other polymer or gel.

The polymer or gel may be functionalized to bind to targeted analytes,such as nucleic acids, proteins, carbohydrates, lipids or otheranalytes. The polymer or gel may be polymerized or gelled via a passivemechanism. The polymer or gel may be stable in alkaline conditions or atelevated temperature. The polymer or gel may have mechanical propertiessimilar to the mechanical properties of the bead. For instance, thepolymer or gel may be of a similar size to the bead. The polymer or gelmay have a mechanical strength (e.g. tensile strength) similar to thatof the bead. The polymer or gel may be of a lower density than an oil.The polymer or gel may be of a density that is roughly similar to thatof a buffer. The polymer or gel may have a tunable pore size. The poresize may be chosen to, for instance, retain denatured nucleic acids. Thepore size may be chosen to maintain diffusive permeability to exogenouschemicals such as sodium hydroxide (NaOH) and/or endogenous chemicalssuch as inhibitors. The polymer or gel may be biocompatible. The polymeror gel may maintain or enhance cell viability. The polymer or gel may bebiochemically compatible. The polymer or gel may be polymerized and/ordepolymerized thermally, chemically, enzymatically, and/or optically.

The polymer may comprise poly(acrylamide-co-acrylic acid) crosslinkedwith disulfide linkages. The preparation of the polymer may comprise atwo-step reaction. In the first activation step,poly(acrylamide-co-acrylic acid) may be exposed to an acylating agent toconvert carboxylic acids to esters. For instance, thepoly(acrylamide-co-acrylic acid) may be exposed to4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM). The polyacrylamide-co-acrylic acid may be exposed to othersalts of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium. Inthe second cross-linking step, the ester formed in the first step may beexposed to a disulfide crosslinking agent. For instance, the ester maybe exposed to cystamine (2,2′-dithiobis(ethylamine)). Following the twosteps, the biological particle may be surrounded by polyacrylamidestrands linked together by disulfide bridges. In this manner, thebiological particle may be encased inside of or comprise a gel or matrix(e.g., polymer matrix) to form a “cell bead.” A cell bead can containbiological particles (e.g., a cell) or macromolecular constituents(e.g., RNA, DNA, proteins, etc.) of biological particles. A cell beadmay include a single cell or multiple cells, or a derivative of thesingle cell or multiple cells. For example, after lysing and washing thecells, inhibitory components from cell lysates can be washed away andthe macromolecular constituents can be bound as cell beads. Systems andmethods disclosed herein can be applicable to both cell beads (and/ordroplets or other partitions) containing biological particles and cellbeads (and/or droplets or other partitions) containing macromolecularconstituents of biological particles.

Encapsulated biological particles can provide certain potentialadvantages of being more storable and more portable than droplet-basedpartitioned biological particles. Furthermore, in some cases, it may bedesirable to allow biological particles to incubate for a select periodof time before analysis, such as in order to characterize changes insuch biological particles over time, either in the presence or absenceof different stimuli. In such cases, encapsulation may allow for longerincubation than partitioning in emulsion droplets, although in somecases, droplet partitioned biological particles may also be incubatedfor different periods of time, e.g., at least 10 seconds, at least 30seconds, at least 1 minute, at least 5 minutes, at least 10 minutes, atleast 30 minutes, at least 1 hour, at least 2 hours, at least 5 hours,or at least 10 hours or more. The encapsulation of biological particlesmay constitute the partitioning of the biological particles into whichother reagents are co-partitioned. Alternatively, or in addition,encapsulated biological particles may be readily deposited into otherpartitions (e.g., droplets) as described above.

A partition may comprise one or more unique identifiers, such asbarcodes. Barcodes may be previously, subsequently or concurrentlydelivered to the partitions that hold the compartmentalized orpartitioned biological particle. For example, barcodes may be injectedinto droplets previous to, subsequent to, or concurrently with dropletgeneration. The delivery of the barcodes to a particular partitionallows for the later attribution of the characteristics of theindividual biological particle to the particular partition. Barcodes maybe delivered, for example on a nucleic acid molecule (e.g., anoligonucleotide), to a partition via any suitable mechanism. Barcodednucleic acid molecules can be delivered to a partition via amicrocapsule. In some cases, barcoded nucleic acid molecules can beinitially associated with the microcapsule and then released from themicrocapsule upon application of a stimulus which allows the nucleicacid molecules to dissociate or to be released from the microcapsule. Amicrocapsule, in some instances, can comprise a bead. Beads aredescribed in further detail below.

FIG. 2 shows an example of a microfluidic channel structure 200 fordelivering barcode carrying beads to droplets. The channel structure 200can include channel segments 201, 202, 204, 206 and 208 communicating ata channel junction 210. In operation, the channel segment 201 maytransport an aqueous fluid 212 that includes a plurality of beads 214(e.g., with nucleic acid molecules, oligonucleotides, molecular tags)along the channel segment 201 into junction 210. The plurality of beads214 may be sourced from a suspension of beads. For example, the channelsegment 201 may be connected to a reservoir comprising an aqueoussuspension of beads 214. The channel segment 202 may transport theaqueous fluid 212 that includes a plurality of biological particles 216along the channel segment 202 into junction 210. The plurality ofbiological particles 216 may be sourced from a suspension of biologicalparticles. For example, the channel segment 202 may be connected to areservoir comprising an aqueous suspension of biological particles 216.In some instances, the aqueous fluid 212 in either the first channelsegment 201 or the second channel segment 202, or in both segments, caninclude one or more reagents, as further described below. A second fluid218 that is immiscible with the aqueous fluid 212 (e.g., oil) can bedelivered to the junction 210 from each of channel segments 204 and 206.Upon meeting of the aqueous fluid 212 from each of channel segments 201and 202 and the second fluid 218 from each of channel segments 204 and206 at the channel junction 210, the aqueous fluid 212 can bepartitioned as discrete droplets 220 in the second fluid 218 and flowaway from the junction 210 along channel segment 208. The channelsegment 208 may deliver the discrete droplets to an outlet reservoirfluidly coupled to the channel segment 208, where they may be harvested.

As an alternative, the channel segments 201 and 202 may meet at anotherjunction upstream of the junction 210. At such junction, beads andbiological particles may form a mixture that is directed along anotherchannel to the junction 210 to yield droplets 220. The mixture mayprovide the beads and biological particles in an alternating fashion,such that, for example, a droplet comprises a single bead and a singlebiological particle.

Beads, biological particles and droplets may flow along channels atsubstantially regular flow profiles (e.g., at regular flow rates). Suchregular flow profiles may permit a droplet to include a single bead anda single biological particle. Such regular flow profiles may permit thedroplets to have an occupancy (e.g., droplets having beads andbiological particles) greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 95%. Such regular flow profiles and devices that maybe used to provide such regular flow profiles are provided in, forexample, U.S. Patent Publication No. 2015/0292988, which is entirelyincorporated herein by reference.

The second fluid 218 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets220.

A discrete droplet that is generated may include an individualbiological particle 216. A discrete droplet that is generated mayinclude a barcode or other reagent carrying bead 214. A discrete dropletgenerated may include both an individual biological particle and abarcode carrying bead, such as droplets 220. In some instances, adiscrete droplet may include more than one individual biologicalparticle or no biological particle. In some instances, a discretedroplet may include more than one bead or no bead. A discrete dropletmay be unoccupied (e.g., no beads, no biological particles).

Beneficially, a discrete droplet partitioning a biological particle anda barcode carrying bead may effectively allow the attribution of thebarcode to macromolecular constituents of the biological particle withinthe partition. The contents of a partition may remain discrete from thecontents of other partitions.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 200 may have other geometries. For example, amicrofluidic channel structure can have more than one channel junctions.For example, a microfluidic channel structure can have 2, 3, 4, or 5channel segments each carrying beads that meet at a channel junction.Fluid may be directed flow along one or more channels or reservoirs viaone or more fluid flow units. A fluid flow unit can comprise compressors(e.g., providing positive pressure), pumps (e.g., providing negativepressure), actuators, and the like to control flow of the fluid. Fluidmay also or otherwise be controlled via applied pressure differentials,centrifugal force, electrokinetic pumping, vacuum, capillary or gravityflow, or the like.

A bead may be porous, non-porous, solid, semi-solid, semi-fluidic,fluidic, and/or a combination thereof. In some instances, a bead may bedissolvable, disruptable, and/or degradable. In some cases, a bead maynot be degradable. In some cases, the bead may be a gel bead. A gel beadmay be a hydrogel bead. A gel bead may be formed from molecularprecursors, such as a polymeric or monomeric species. A semi-solid beadmay be a liposomal bead. Solid beads may comprise metals including ironoxide, gold, and silver. In some cases, the bead may be a silica bead.In some cases, the bead can be rigid. In other cases, the bead may beflexible and/or compressible.

A bead may be of any suitable shape. Examples of bead shapes include,but are not limited to, spherical, non-spherical, oval, oblong,amorphous, circular, cylindrical, and variations thereof.

Beads may be of uniform size or heterogeneous size. In some cases, thediameter of a bead may be at least about 1 micrometers (μm), 5 μm, 10μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 250μm, 500 μm, 1 mm, or greater. In some cases, a bead may have a diameterof less than about 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm,70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, 1 mm, or less. In somecases, a bead may have a diameter in the range of about 40-75 μm, 30-75μm, 20-75 μm, 40-85 μm, 40-95 μm, 20-100 μm, 10-100 μm, 1-100 μm, 20-250μm, or 20-500 μm.

In certain aspects, beads can be provided as a population or pluralityof beads having a relatively monodisperse size distribution. Where itmay be desirable to provide relatively consistent amounts of reagentswithin partitions, maintaining relatively consistent beadcharacteristics, such as size, can contribute to the overallconsistency. In particular, the beads described herein may have sizedistributions that have a coefficient of variation in theircross-sectional dimensions of less than 50%, less than 40%, less than30%, less than 20%, and in some cases less than 15%, less than 10%, lessthan 5%, or less.

A bead may comprise natural and/or synthetic materials. For example, abead can comprise a natural polymer, a synthetic polymer or both naturaland synthetic polymers. Examples of natural polymers include proteinsand sugars such as deoxyribonucleic acid, rubber, cellulose, starch(e.g., amylose, amylopectin), proteins, enzymes, polysaccharides, silks,polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan,ispaghula, acacia, agar, gelatin, shellac, sterculia gum, xanthan gum,Corn sugar gum, guar gum, gum karaya, agarose, alginic acid, alginate,or natural polymers thereof. Examples of synthetic polymers includeacrylics, nylons, silicones, spandex, viscose rayon, polycarboxylicacids, polyvinyl acetate, polyacrylamide, polyacrylate, polyethyleneglycol, polyurethanes, polylactic acid, silica, polystyrene,polyacrylonitrile, polybutadiene, polycarbonate, polyethylene,polyethylene terephthalate, poly(chlorotrifluoroethylene), poly(ethyleneoxide), poly(ethylene terephthalate), polyethylene, polyisobutylene,poly(methyl methacrylate), poly(oxymethylene), polyformaldehyde,polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinylacetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidenedichloride), poly(vinylidene difluoride), poly(vinyl fluoride) and/orcombinations (e.g., co-polymers) thereof. Beads may also be formed frommaterials other than polymers, including lipids, micelles, ceramics,glass-ceramics, material composites, metals, other inorganic materials,and others.

In some instances, the bead may contain molecular precursors (e.g.,monomers or polymers), which may form a polymer network viapolymerization of the molecular precursors. In some cases, a precursormay be an already polymerized species capable of undergoing furtherpolymerization via, for example, a chemical cross-linkage In some cases,a precursor can comprise one or more of an acrylamide or amethacrylamide monomer, oligomer, or polymer. In some cases, the beadmay comprise prepolymers, which are oligomers capable of furtherpolymerization. For example, polyurethane beads may be prepared usingprepolymers. In some cases, the bead may contain individual polymersthat may be further polymerized together. In some cases, beads may begenerated via polymerization of different precursors, such that theycomprise mixed polymers, co-polymers, and/or block co-polymers. In somecases, the bead may comprise covalent or ionic bonds between polymericprecursors (e.g., monomers, oligomers, linear polymers), nucleic acidmolecules (e.g., oligonucleotides), primers, and other entities. In somecases, the covalent bonds can be carbon-carbon bonds or thioether bonds.

Cross-linking may be permanent or reversible, depending upon theparticular cross-linker used. Reversible cross-linking may allow for thepolymer to linearize or dissociate under appropriate conditions. In somecases, reversible cross-linking may also allow for reversible attachmentof a material bound to the surface of a bead. In some cases, across-linker may form disulfide linkages. In some cases, the chemicalcross-linker forming disulfide linkages may be cystamine or a modifiedcystamine.

In some cases, disulfide linkages can be formed between molecularprecursor units (e.g., monomers, oligomers, or linear polymers) orprecursors incorporated into a bead and nucleic acid molecules (e.g.,oligonucleotides). Cystamine (including modified cystamines), forexample, is an organic agent comprising a disulfide bond that may beused as a crosslinker agent between individual monomeric or polymericprecursors of a bead. Polyacrylamide may be polymerized in the presenceof cystamine or a species comprising cystamine (e.g., a modifiedcystamine) to generate polyacrylamide gel beads comprising disulfidelinkages (e.g., chemically degradable beads comprisingchemically-reducible cross-linkers). The disulfide linkages may permitthe bead to be degraded (or dissolved) upon exposure of the bead to areducing agent.

In some cases, chitosan, a linear polysaccharide polymer, may becrosslinked with glutaraldehyde via hydrophilic chains to form a bead.Crosslinking of chitosan polymers may be achieved by chemical reactionsthat are initiated by heat, pressure, change in pH, and/or radiation.

In some cases, a bead may comprise an acrydite moiety, which in certainaspects may be used to attach one or more nucleic acid molecules (e.g.,barcode sequence, barcoded nucleic acid molecule, barcodedoligonucleotide, primer, or other oligonucleotide) to the bead. In somecases, an acrydite moiety can refer to an acrydite analogue generatedfrom the reaction of acrydite with one or more species, such as, thereaction of acrydite with other monomers and cross-linkers during apolymerization reaction. Acrydite moieties may be modified to formchemical bonds with a species to be attached, such as a nucleic acidmolecule (e.g., barcode sequence, barcoded nucleic acid molecule,barcoded oligonucleotide, primer, or other oligonucleotide). Acryditemoieties may be modified with thiol groups capable of forming adisulfide bond or may be modified with groups already comprising adisulfide bond. The thiol or disulfide (via disulfide exchange) may beused as an anchor point for a species to be attached or another part ofthe acrydite moiety may be used for attachment. In some cases,attachment can be reversible, such that when the disulfide bond isbroken (e.g., in the presence of a reducing agent), the attached speciesis released from the bead. In other cases, an acrydite moiety cancomprise a reactive hydroxyl group that may be used for attachment.

Functionalization of beads for attachment of nucleic acid molecules(e.g., oligonucleotides) may be achieved through a wide range ofdifferent approaches, including activation of chemical groups within apolymer, incorporation of active or activatable functional groups in thepolymer structure, or attachment at the pre-polymer or monomer stage inbead production.

For example, precursors (e.g., monomers, cross-linkers) that arepolymerized to form a bead may comprise acrydite moieties, such thatwhen a bead is generated, the bead also comprises acrydite moieties. Theacrydite moieties can be attached to a nucleic acid molecule (e.g.,oligonucleotide), which may include a priming sequence (e.g., a primerfor amplifying target nucleic acids, random primer, primer sequence formessenger RNA) and/or a one or more barcode sequences. The one morebarcode sequences may include sequences that are the same for allnucleic acid molecules coupled to a given bead and/or sequences that aredifferent across all nucleic acid molecules coupled to the given bead.The nucleic acid molecule may be incorporated into the bead.

In some cases, the nucleic acid molecule can comprise a functionalsequence, for example, for attachment to a sequencing flow cell, suchas, for example, a P5 sequence for Illumina® sequencing. In some cases,the nucleic acid molecule or derivative thereof (e.g., oligonucleotideor polynucleotide generated from the nucleic acid molecule) can compriseanother functional sequence, such as, for example, a P7 sequence forattachment to a sequencing flow cell for Illumina sequencing. In somecases, the nucleic acid molecule can comprise a barcode sequence. Insome cases, the primer can further comprise a unique molecularidentifier (UMI). In some cases, the primer can comprise an R1 primersequence for Illumina sequencing. In some cases, the primer can comprisean R2 primer sequence for Illumina sequencing. Examples of such nucleicacid molecules (e.g., oligonucleotides, polynucleotides, etc.) and usesthereof, as may be used with compositions, devices, methods and systemsof the present disclosure, are provided in U.S. Patent Pub. Nos.2014/0378345 and 2015/0376609, each of which is entirely incorporatedherein by reference.

In some cases, precursors comprising a functional group that is reactiveor capable of being activated such that it becomes reactive can bepolymerized with other precursors to generate gel beads comprising theactivated or activatable functional group. The functional group may thenbe used to attach additional species (e.g., disulfide linkers, primers,other oligonucleotides, etc.) to the gel beads. For example, someprecursors comprising a carboxylic acid (COOH) group can co-polymerizewith other precursors to form a gel bead that also comprises a COOHfunctional group. In some cases, acrylic acid (a species comprising freeCOOH groups), acrylamide, and bis(acryloyl)cystamine can beco-polymerized together to generate a gel bead comprising free COOHgroups. The COOH groups of the gel bead can be activated (e.g., via1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-Hydroxysuccinimide (NHS) or4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM)) such that they are reactive (e.g., reactive to amine functionalgroups where EDC/NHS or DMTMM are used for activation). The activatedCOOH groups can then react with an appropriate species (e.g., a speciescomprising an amine functional group where the carboxylic acid groupsare activated to be reactive with an amine functional group) comprisinga moiety to be linked to the bead.

Beads comprising disulfide linkages in their polymeric network may befunctionalized with additional species via reduction of some of thedisulfide linkages to free thiols. The disulfide linkages may be reducedvia, for example, the action of a reducing agent (e.g., DTT, TCEP, etc.)to generate free thiol groups, without dissolution of the bead. Freethiols of the beads can then react with free thiols of a species or aspecies comprising another disulfide bond (e.g., via thiol-disulfideexchange) such that the species can be linked to the beads (e.g., via agenerated disulfide bond). In some cases, free thiols of the beads mayreact with any other suitable group. For example, free thiols of thebeads may react with species comprising an acrydite moiety. The freethiol groups of the beads can react with the acrydite via Michaeladdition chemistry, such that the species comprising the acrydite islinked to the bead. In some cases, uncontrolled reactions can beprevented by inclusion of a thiol capping agent such as N-ethylmaleimideor iodoacetate.

Activation of disulfide linkages within a bead can be controlled suchthat only a small number of disulfide linkages are activated. Controlmay be exerted, for example, by controlling the concentration of areducing agent used to generate free thiol groups and/or concentrationof reagents used to form disulfide bonds in bead polymerization. In somecases, a low concentration (e.g., molecules of reducing agent:gel beadratios of less than or equal to about 1:100,000,000,000, less than orequal to about 1:10,000,000,000, less than or equal to about1:1,000,000,000, less than or equal to about 1:100,000,000, less than orequal to about 1:10,000,000, less than or equal to about 1:1,000,000,less than or equal to about 1:100,000, less than or equal to about1:10,000) of reducing agent may be used for reduction. Controlling thenumber of disulfide linkages that are reduced to free thiols may beuseful in ensuring bead structural integrity during functionalization.In some cases, optically-active agents, such as fluorescent dyes may becoupled to beads via free thiol groups of the beads and used to quantifythe number of free thiols present in a bead and/or track a bead.

In some cases, addition of moieties to a gel bead after gel beadformation may be advantageous. For example, addition of anoligonucleotide (e.g., barcoded oligonucleotide) after gel beadformation may avoid loss of the species during chain transfertermination that can occur during polymerization. Moreover, smallerprecursors (e.g., monomers or cross linkers that do not comprise sidechain groups and linked moieties) may be used for polymerization and canbe minimally hindered from growing chain ends due to viscous effects. Insome cases, functionalization after gel bead synthesis can minimizeexposure of species (e.g., oligonucleotides) to be loaded withpotentially damaging agents (e.g., free radicals) and/or chemicalenvironments. In some cases, the generated gel may possess an uppercritical solution temperature (UCST) that can permit temperature drivenswelling and collapse of a bead. Such functionality may aid inoligonucleotide (e.g., a primer) infiltration into the bead duringsubsequent functionalization of the bead with the oligonucleotide.Post-production functionalization may also be useful in controllingloading ratios of species in beads, such that, for example, thevariability in loading ratio is minimized Species loading may also beperformed in a batch process such that a plurality of beads can befunctionalized with the species in a single batch.

A bead injected or otherwise introduced into a partition may comprisereleasably, cleavably, or reversibly attached barcodes. A bead injectedor otherwise introduced into a partition may comprise activatablebarcodes. A bead injected or otherwise introduced into a partition maybe degradable, disruptable, or dissolvable beads.

Barcodes can be releasably, cleavably or reversibly attached to thebeads such that barcodes can be released or be releasable throughcleavage of a linkage between the barcode molecule and the bead, orreleased through degradation of the underlying bead itself, allowing thebarcodes to be accessed or be accessible by other reagents, or both. Innon-limiting examples, cleavage may be achieved through reduction ofdi-sulfide bonds, use of restriction enzymes, photo-activated cleavage,or cleavage via other types of stimuli (e.g., chemical, thermal, pH,enzymatic, etc.) and/or reactions, such as described elsewhere herein.Releasable barcodes may sometimes be referred to as being activatable,in that they are available for reaction once released. Thus, forexample, an activatable barcode may be activated by releasing thebarcode from a bead (or other suitable type of partition describedherein). Other activatable configurations are also envisioned in thecontext of the described methods and systems.

In addition to, or as an alternative to the cleavable linkages betweenthe beads and the associated molecules, such as barcode containingnucleic acid molecules (e.g., barcoded oligonucleotides), the beads maybe degradable, disruptable, or dissolvable spontaneously or uponexposure to one or more stimuli (e.g., temperature changes, pH changes,exposure to particular chemical species or phase, exposure to light,reducing agent, etc.). In some cases, a bead may be dissolvable, suchthat material components of the beads are solubilized when exposed to aparticular chemical species or an environmental change, such as a changetemperature or a change in pH. In some cases, a gel bead can be degradedor dissolved at elevated temperature and/or in basic conditions. In somecases, a bead may be thermally degradable such that when the bead isexposed to an appropriate change in temperature (e.g., heat), the beaddegrades. Degradation or dissolution of a bead bound to a species (e.g.,a nucleic acid molecule, e.g., barcoded oligonucleotide) may result inrelease of the species from the bead.

As will be appreciated from the above disclosure, the degradation of abead may refer to the disassociation of a bound or entrained speciesfrom a bead, both with and without structurally degrading the physicalbead itself. For example, the degradation of the bead may involvecleavage of a cleavable linkage via one or more species and/or methodsdescribed elsewhere herein. In another example, entrained species may bereleased from beads through osmotic pressure differences due to, forexample, changing chemical environments. By way of example, alterationof bead pore sizes due to osmotic pressure differences can generallyoccur without structural degradation of the bead itself. In some cases,an increase in pore size due to osmotic swelling of a bead can permitthe release of entrained species within the bead. In other cases,osmotic shrinking of a bead may cause a bead to better retain anentrained species due to pore size contraction.

A degradable bead may be introduced into a partition, such as a dropletof an emulsion or a well, such that the bead degrades within thepartition and any associated species (e.g., oligonucleotides) arereleased within the droplet when the appropriate stimulus is applied.The free species (e.g., oligonucleotides, nucleic acid molecules) mayinteract with other reagents contained in the partition. For example, apolyacrylamide bead comprising cystamine and linked, via a disulfidebond, to a barcode sequence, may be combined with a reducing agentwithin a droplet of a water-in-oil emulsion. Within the droplet, thereducing agent can break the various disulfide bonds, resulting in beaddegradation and release of the barcode sequence into the aqueous, innerenvironment of the droplet. In another example, heating of a dropletcomprising a bead-bound barcode sequence in basic solution may alsoresult in bead degradation and release of the attached barcode sequenceinto the aqueous, inner environment of the droplet.

Any suitable number of molecular tag molecules (e.g., primer, barcodedoligonucleotide) can be associated with a bead such that, upon releasefrom the bead, the molecular tag molecules (e.g., primer, e.g., barcodedoligonucleotide) are present in the partition at a pre-definedconcentration. Such pre-defined concentration may be selected tofacilitate certain reactions for generating a sequencing library, e.g.,amplification, within the partition. In some cases, the pre-definedconcentration of the primer can be limited by the process of producingnucleic acid molecule (e.g., oligonucleotide) bearing beads.

In some cases, beads can be non-covalently loaded with one or morereagents. The beads can be non-covalently loaded by, for instance,subjecting the beads to conditions sufficient to swell the beads,allowing sufficient time for the reagents to diffuse into the interiorsof the beads, and subjecting the beads to conditions sufficient tode-swell the beads. The swelling of the beads may be accomplished, forinstance, by placing the beads in a thermodynamically favorable solvent,subjecting the beads to a higher or lower temperature, subjecting thebeads to a higher or lower ion concentration, and/or subjecting thebeads to an electric field. The swelling of the beads may beaccomplished by various swelling methods. The de-swelling of the beadsmay be accomplished, for instance, by transferring the beads in athermodynamically unfavorable solvent, subjecting the beads to lower orhigh temperatures, subjecting the beads to a lower or higher ionconcentration, and/or removing an electric field. The de-swelling of thebeads may be accomplished by various de-swelling methods. Transferringthe beads may cause pores in the bead to shrink. The shrinking may thenhinder reagents within the beads from diffusing out of the interiors ofthe beads. The hindrance may be due to steric interactions between thereagents and the interiors of the beads. The transfer may beaccomplished microfluidically. For instance, the transfer may beachieved by moving the beads from one co-flowing solvent stream to adifferent co-flowing solvent stream. The swellability and/or pore sizeof the beads may be adjusted by changing the polymer composition of thebead.

In some cases, an acrydite moiety linked to a precursor, another specieslinked to a precursor, or a precursor itself can comprise a labile bond,such as chemically, thermally, or photo-sensitive bond e.g., disulfidebond, UV sensitive bond, or the like. Once acrydite moieties or othermoieties comprising a labile bond are incorporated into a bead, the beadmay also comprise the labile bond. The labile bond may be, for example,useful in reversibly linking (e.g., covalently linking) species (e.g.,barcodes, primers, etc.) to a bead. In some cases, a thermally labilebond may include a nucleic acid hybridization based attachment, e.g.,where an oligonucleotide is hybridized to a complementary sequence thatis attached to the bead, such that thermal melting of the hybridreleases the oligonucleotide, e.g., a barcode containing sequence, fromthe bead or microcapsule.

The addition of multiple types of labile bonds to a gel bead may resultin the generation of a bead capable of responding to varied stimuli.Each type of labile bond may be sensitive to an associated stimulus(e.g., chemical stimulus, light, temperature, enzymatic, etc.) such thatrelease of species attached to a bead via each labile bond may becontrolled by the application of the appropriate stimulus. Suchfunctionality may be useful in controlled release of species from a gelbead. In some cases, another species comprising a labile bond may belinked to a gel bead after gel bead formation via, for example, anactivated functional group of the gel bead as described above. As willbe appreciated, barcodes that are releasably, cleavably or reversiblyattached to the beads described herein include barcodes that arereleased or releasable through cleavage of a linkage between the barcodemolecule and the bead, or that are released through degradation of theunderlying bead itself, allowing the barcodes to be accessed oraccessible by other reagents, or both.

The barcodes that are releasable as described herein may sometimes bereferred to as being activatable, in that they are available forreaction once released. Thus, for example, an activatable barcode may beactivated by releasing the barcode from a bead (or other suitable typeof partition described herein). Other activatable configurations arealso envisioned in the context of the described methods and systems.

In addition to thermally cleavable bonds, disulfide bonds and UVsensitive bonds, other non-limiting examples of labile bonds that may becoupled to a precursor or bead include an ester linkage (e.g., cleavablewith an acid, a base, or hydroxylamine), a carbamate linkage (e.g.,cleavable with diethylenetriamine “DETA”), a vicinal diol linkage (e.g.,cleavable via sodium periodate), a Diels-Alder linkage (e.g., cleavablevia heat), a sulfone linkage (e.g., cleavable via a base), a silyl etherlinkage (e.g., cleavable via an acid), a glycosidic linkage (e.g.,cleavable via an amylase), a peptide linkage (e.g., cleavable via aprotease), or a phosphodiester linkage (e.g., cleavable via a nuclease(e.g., DNase)). A bond may be cleavable via other nucleic acid moleculetargeting enzymes, such as restriction enzymes (e.g., restrictionendonucleases), as described further below.

Species may be encapsulated in beads during bead generation (e.g.,during polymerization of precursors). Such species may or may notparticipate in polymerization. Such species may be entered intopolymerization reaction mixtures such that generated beads comprise thespecies upon bead formation. In some cases, such species may be added tothe gel beads after formation. Such species may include, for example,nucleic acid molecules (e.g., oligonucleotides), reagents for a nucleicacid amplification reaction (e.g., primers, polymerases, dNTPs,co-factors (e.g., ionic co-factors), buffers) including those describedherein, reagents for enzymatic reactions (e.g., enzymes, co-factors,substrates, buffers), reagents for nucleic acid modification reactionssuch as polymerization, ligation, or digestion, and/or reagents fortemplate preparation (e.g., tagmentation) for one or more sequencingplatforms (e.g., NEXTERA® for ILLUMINA®). Such species may include oneor more enzymes described herein, including without limitation,polymerase, reverse transcriptase, restriction enzymes (e.g.,endonuclease), transposase, ligase, proteinase K, DNase, etc. Suchspecies may include one or more reagents described elsewhere herein(e.g., lysis agents, inhibitors, inactivating agents, chelating agents,stimulus). Trapping of such species may be controlled by the polymernetwork density generated during polymerization of precursors, controlof ionic charge within the gel bead (e.g., via ionic species linked topolymerized species), or by the release of other species. Encapsulatedspecies may be released from a bead upon bead degradation and/or byapplication of a stimulus capable of releasing the species from thebead. Alternatively, or in addition, species may be partitioned in apartition (e.g., droplet) during or subsequent to partition formation.Such species may include, without limitation, the abovementioned speciesthat may also be encapsulated in a bead.

A degradable bead may comprise one or more species with a labile bondsuch that, when the bead/species is exposed to the appropriate stimuli,the bond is broken, and the bead degrades. The labile bond may be achemical bond (e.g., covalent bond, ionic bond) or may be another typeof physical interaction (e.g., van der Waals interactions, dipole-dipoleinteractions, etc.). In some cases, a crosslinker used to generate abead may comprise a labile bond. Upon exposure to the appropriateconditions, the labile bond can be broken, and the bead degraded. Forexample, upon exposure of a polyacrylamide gel bead comprising cystaminecrosslinkers to a reducing agent, the disulfide bonds of the cystaminecan be broken and the bead degraded.

A degradable bead may be useful in more quickly releasing an attachedspecies (e.g., a nucleic acid molecule, a barcode sequence, a primer,etc.) from the bead when the appropriate stimulus is applied to the beadas compared to a bead that does not degrade. For example, for a speciesbound to an inner surface of a porous bead or in the case of anencapsulated species, the species may have greater mobility andaccessibility to other species in solution upon degradation of the bead.In some cases, a species may also be attached to a degradable bead via adegradable linker (e.g., disulfide linker). The degradable linker mayrespond to the same stimuli as the degradable bead or the two degradablespecies may respond to different stimuli. For example, a barcodesequence may be attached, via a disulfide bond, to a polyacrylamide beadcomprising cystamine Upon exposure of the barcoded-bead to a reducingagent, the bead degrades, and the barcode sequence is released uponbreakage of both the disulfide linkage between the barcode sequence andthe bead and the disulfide linkages of the cystamine in the bead.

As will be appreciated from the above disclosure, while referred to asdegradation of a bead, in many instances as noted above, thatdegradation may refer to the disassociation of a bound or entrainedspecies from a bead, both with and without structurally degrading thephysical bead itself. For example, entrained species may be releasedfrom beads through osmotic pressure differences due to, for example,changing chemical environments. By way of example, alteration of beadpore sizes due to osmotic pressure differences can generally occurwithout structural degradation of the bead itself. In some cases, anincrease in pore size due to osmotic swelling of a bead can permit therelease of entrained species within the bead. In other cases, osmoticshrinking of a bead may cause a bead to better retain an entrainedspecies due to pore size contraction.

Where degradable beads are provided, it may be beneficial to avoidexposing such beads to the stimulus or stimuli that cause suchdegradation prior to a given time, in order to, for example, avoidpremature bead degradation and issues that arise from such degradation,including for example poor flow characteristics and aggregation. By wayof example, where beads comprise reducible cross-linking groups, such asdisulfide groups, it will be desirable to avoid contacting such beadswith reducing agents, e.g., DTT or other disulfide cleaving reagents. Insuch cases, treatment to the beads described herein will, in some casesbe provided free of reducing agents, such as DTT. Because reducingagents are often provided in commercial enzyme preparations, it may bedesirable to provide reducing agent free (or DTT free) enzymepreparations in treating the beads described herein. Examples of suchenzymes include, e.g., polymerase enzyme preparations, reversetranscriptase enzyme preparations, ligase enzyme preparations, as wellas many other enzyme preparations that may be used to treat the beadsdescribed herein. The terms “reducing agent free” or “DTT free”preparations can refer to a preparation having less than about 1/10th,less than about 1/50th, or even less than about 1/100th of the lowerranges for such materials used in degrading the beads. For example, forDTT, the reducing agent free preparation can have less than about 0.01millimolar (mM), 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or even lessthan about 0.0001 mM DTT. In many cases, the amount of DTT can beundetectable.

Numerous chemical triggers may be used to trigger the degradation ofbeads. Examples of these chemical changes may include but are notlimited to pH-mediated changes to the integrity of a component withinthe bead, degradation of a component of a bead via cleavage ofcross-linked bonds, and depolymerization of a component of a bead.

In some embodiments, a bead may be formed from materials that comprisedegradable chemical crosslinkers, such as BAC or cystamine Degradationof such degradable crosslinkers may be accomplished through a number ofmechanisms. In some examples, a bead may be contacted with a chemicaldegrading agent that may induce oxidation, reduction or other chemicalchanges. For example, a chemical degrading agent may be a reducingagent, such as dithiothreitol (DTT). Additional examples of reducingagents may include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), orcombinations thereof. A reducing agent may degrade the disulfide bondsformed between gel precursors forming the bead, and thus, degrade thebead. In other cases, a change in pH of a solution, such as an increasein pH, may trigger degradation of a bead. In other cases, exposure to anaqueous solution, such as water, may trigger hydrolytic degradation, andthus degradation of the bead.

Beads may also be induced to release their contents upon the applicationof a thermal stimulus. A change in temperature can cause a variety ofchanges to a bead. For example, heat can cause a solid bead to liquefy.A change in heat may cause melting of a bead such that a portion of thebead degrades. In other cases, heat may increase the internal pressureof the bead components such that the bead ruptures or explodes. Heat mayalso act upon heat-sensitive polymers used as materials to constructbeads.

Any suitable agent may degrade beads. In some embodiments, changes intemperature or pH may be used to degrade thermo-sensitive orpH-sensitive bonds within beads. In some embodiments, chemical degradingagents may be used to degrade chemical bonds within beads by oxidation,reduction or other chemical changes. For example, a chemical degradingagent may be a reducing agent, such as DTT, wherein DTT may degrade thedisulfide bonds formed between a crosslinker and gel precursors, thusdegrading the bead. In some embodiments, a reducing agent may be addedto degrade the bead, which may or may not cause the bead to release itscontents. Examples of reducing agents may include dithiothreitol (DTT),β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamineor DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinationsthereof. The reducing agent may be present at a concentration of about0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM. The reducing agent may be present ata concentration of at least about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, orgreater than 10 mM. The reducing agent may be present at concentrationof at most about 10 mM, 5 mM, 1 mM, 0.5 mM, 0.1 mM, or less.

Any suitable number of molecular tag molecules (e.g., primer, barcodedoligonucleotide) can be associated with a bead such that, upon releasefrom the bead, the molecular tag molecules (e.g., primer, e.g., barcodedoligonucleotide) are present in the partition at a pre-definedconcentration. Such pre-defined concentration may be selected tofacilitate certain reactions for generating a sequencing library, e.g.,amplification, within the partition. In some cases, the pre-definedconcentration of the primer can be limited by the process of producingoligonucleotide bearing beads.

Although FIG. 1 and FIG. 2 have been described in terms of providingsubstantially singly occupied partitions, above, in certain cases, itmay be desirable to provide multiply occupied partitions, e.g.,containing two, three, four or more cells and/or microcapsules (e.g.,beads) comprising barcoded nucleic acid molecules (e.g.,oligonucleotides) within a single partition. Accordingly, as notedabove, the flow characteristics of the biological particle and/or beadcontaining fluids and partitioning fluids may be controlled to providefor such multiply occupied partitions. In particular, the flowparameters may be controlled to provide a given occupancy rate atgreater than about 50% of the partitions, greater than about 75%, and insome cases greater than about 80%, 90%, 95%, or higher.

In some cases, additional microcapsules can be used to deliveradditional reagents to a partition. In such cases, it may beadvantageous to introduce different beads into a common channel ordroplet generation junction, from different bead sources (e.g.,containing different associated reagents) through different channelinlets into such common channel or droplet generation junction (e.g.,junction 210). In such cases, the flow and frequency of the differentbeads into the channel or junction may be controlled to provide for acertain ratio of microcapsules from each source, while ensuring a givenpairing or combination of such beads into a partition with a givennumber of biological particles (e.g., one biological particle and onebead per partition).

The partitions described herein may comprise small volumes, for example,less than about 10 microliters (μL), 5 μL, 1 μL, 900 picoliters (pL),800 pL, 700 pL, 600 pL, 500 pL, 400pL, 300 pL, 200 pL, 100 pL, 50 pL, 20pL, 10 pL, 1 pL, 500 nanoliters (nL), 100 nL, 50 nL, or less.

For example, in the case of droplet based partitions, the droplets mayhave overall volumes that are less than about 1000 pL, 900 pL, 800 pL,700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL, 20 pL, 10pL, 1 pL, or less. Where co-partitioned with microcapsules, it will beappreciated that the sample fluid volume, e.g., including co-partitionedbiological particles and/or beads, within the partitions may be lessthan about 90% of the above described volumes, less than about 80%, lessthan about 70%, less than about 60%, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, or less than about10% of the above described volumes.

As is described elsewhere herein, partitioning species may generate apopulation or plurality of partitions. In such cases, any suitablenumber of partitions can be generated or otherwise provided. Forexample, at least about 1,000 partitions, at least about 5,000partitions, at least about 10,000 partitions, at least about 50,000partitions, at least about 100,000 partitions, at least about 500,000partitions, at least about 1,000,000 partitions, at least about5,000,000 partitions at least about 10,000,000 partitions, at leastabout 50,000,000 partitions, at least about 100,000,000 partitions, atleast about 500,000,000 partitions, at least about 1,000,000,000partitions, or more partitions can be generated or otherwise provided.Moreover, the plurality of partitions may comprise both unoccupiedpartitions (e.g., empty partitions) and occupied partitions.

In accordance with certain aspects, biological particles may bepartitioned along with lysis reagents in order to release the contentsof the biological particles within the partition. In such cases, thelysis agents can be contacted with the biological particle suspensionconcurrently with, or immediately prior to, the introduction of thebiological particles into the partitioning junction/droplet generationzone (e.g., junction 210), such as through an additional channel orchannels upstream of the channel junction. In accordance with otheraspects, additionally or alternatively, biological particles may bepartitioned along with other reagents, as will be described furtherbelow.

FIG. 3 shows an example of a microfluidic channel structure 300 forco-partitioning biological particles and reagents. The channel structure300 can include channel segments 301, 302, 304, 306 and 308. Channelsegments 301 and 302 communicate at a first channel junction 309.Channel segments 302, 304, 306, and 308 communicate at a second channeljunction 310.

In an example operation, the channel segment 301 may transport anaqueous fluid 312 that includes a plurality of biological particles 314along the channel segment 301 into the second junction 310. As analternative or in addition to, channel segment 301 may transport beads(e.g., gel beads). The beads may comprise barcode molecules.

For example, the channel segment 301 may be connected to a reservoircomprising an aqueous suspension of biological particles 314. Upstreamof, and immediately prior to reaching, the second junction 310, thechannel segment 301 may meet the channel segment 302 at the firstjunction 309. The channel segment 302 may transport a plurality ofreagents 315 (e.g., lysis agents) suspended in the aqueous fluid 312along the channel segment 302 into the first junction 309. For example,the channel segment 302 may be connected to a reservoir comprising thereagents 315. After the first junction 309, the aqueous fluid 312 in thechannel segment 301 can carry both the biological particles 314 and thereagents 315 towards the second junction 310. In some instances, theaqueous fluid 312 in the channel segment 301 can include one or morereagents, which can be the same or different reagents as the reagents315. A second fluid 316 that is immiscible with the aqueous fluid 312(e.g., oil) can be delivered to the second junction 310 from each ofchannel segments 304 and 306. Upon meeting of the aqueous fluid 312 fromthe channel segment 301 and the second fluid 316 from each of channelsegments 304 and 306 at the second channel junction 310, the aqueousfluid 312 can be partitioned as discrete droplets 318 in the secondfluid 316 and flow away from the second junction 310 along channelsegment 308. The channel segment 308 may deliver the discrete droplets318 to an outlet reservoir fluidly coupled to the channel segment 308,where they may be harvested.

The second fluid 316 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets318.

A discrete droplet generated may include an individual biologicalparticle 314 and/or one or more reagents 315. In some instances, adiscrete droplet generated may include a barcode carrying bead (notshown), such as via other microfluidics structures described elsewhereherein. In some instances, a discrete droplet may be unoccupied (e.g.,no reagents, no biological particles).

Beneficially, when lysis reagents and biological particles areco-partitioned, the lysis reagents can facilitate the release of thecontents of the biological particles within the partition. The contentsreleased in a partition may remain discrete from the contents of otherpartitions.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 300 may have other geometries. For example, amicrofluidic channel structure can have more than two channel junctions.For example, a microfluidic channel structure can have 2, 3, 4, 5channel segments or more each carrying the same or different types ofbeads, reagents, and/or biological particles that meet at a channeljunction. Fluid flow in each channel segment may be controlled tocontrol the partitioning of the different elements into droplets. Fluidmay be directed flow along one or more channels or reservoirs via one ormore fluid flow units. A fluid flow unit can comprise compressors (e.g.,providing positive pressure), pumps (e.g., providing negative pressure),actuators, and the like to control flow of the fluid. Fluid may also orotherwise be controlled via applied pressure differentials, centrifugalforce, electrokinetic pumping, vacuum, capillary or gravity flow, or thelike.

Examples of lysis agents include bioactive reagents, such as lysisenzymes that are used for lysis of different cell types, e.g., grampositive or negative bacteria, plants, yeast, mammalian, etc., such aslysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase,and a variety of other lysis enzymes available from, e.g.,Sigma-Aldrich, Inc. (St Louis, Miss.), as well as other commerciallyavailable lysis enzymes. Other lysis agents may additionally oralternatively be co-partitioned with the biological particles to causethe release of the biological particles' contents into the partitions.For example, in some cases, surfactant-based lysis solutions may be usedto lyse cells, although these may be less desirable for emulsion basedsystems where the surfactants can interfere with stable emulsions. Insome cases, lysis solutions may include non-ionic surfactants such as,for example, TritonX-100 and Tween 20. In some cases, lysis solutionsmay include ionic surfactants such as, for example, sarcosyl and sodiumdodecyl sulfate (SDS). Electroporation, thermal, acoustic or mechanicalcellular disruption may also be used in certain cases, e.g.,non-emulsion based partitioning such as encapsulation of biologicalparticles that may be in addition to or in place of dropletpartitioning, where any pore size of the encapsulate is sufficientlysmall to retain nucleic acid fragments of a given size, followingcellular disruption.

In addition to the lysis agents co-partitioned with the biologicalparticles described above, other reagents can also be co-partitionedwith the biological particles, including, for example, DNase and RNaseinactivating agents or inhibitors, such as proteinase K, chelatingagents, such as EDTA, and other reagents employed in removing orotherwise reducing negative activity or impact of different cell lysatecomponents on subsequent processing of nucleic acids. In addition, inthe case of encapsulated biological particles, the biological particlesmay be exposed to an appropriate stimulus to release the biologicalparticles or their contents from a co-partitioned microcapsule. Forexample, in some cases, a chemical stimulus may be co-partitioned alongwith an encapsulated biological particle to allow for the degradation ofthe microcapsule and release of the cell or its contents into the largerpartition. In some cases, this stimulus may be the same as the stimulusdescribed elsewhere herein for release of nucleic acid molecules (e.g.,oligonucleotides) from their respective microcapsule (e.g., bead). Inalternative aspects, this may be a different and non-overlappingstimulus, in order to allow an encapsulated biological particle to bereleased into a partition at a different time from the release ofnucleic acid molecules into the same partition.

Additional reagents may also be co-partitioned with the biologicalparticles, such as endonucleases to fragment a biological particle'sDNA, DNA polymerase enzymes and dNTPs used to amplify the biologicalparticle's nucleic acid fragments and to attach the barcode moleculartags to the amplified fragments. Other enzymes may be co-partitioned,including without limitation, polymerase, transposase, ligase,proteinase K, DNase, etc. Additional reagents may also include reversetranscriptase enzymes, including enzymes with terminal transferaseactivity, primers and oligonucleotides, and switch oligonucleotides(also referred to herein as “switch oligos” or “template switchingoligonucleotides”) which can be used for template switching. In somecases, template switching can be used to increase the length of a cDNA.In some cases, template switching can be used to append a predefinednucleic acid sequence to the cDNA. In an example of template switching,cDNA can be generated from reverse transcription of a template, e.g.,cellular mRNA, where a reverse transcriptase with terminal transferaseactivity can add additional nucleotides, e.g., polyC, to the cDNA in atemplate independent manner. Switch oligos can include sequencescomplementary to the additional nucleotides, e.g., polyG. The additionalnucleotides (e.g., polyC) on the cDNA can hybridize to the additionalnucleotides (e.g., polyG) on the switch oligo, whereby the switch oligocan be used by the reverse transcriptase as template to further extendthe cDNA. Template switching oligonucleotides may comprise ahybridization region and a template region. The hybridization region cancomprise any sequence capable of hybridizing to the target. In somecases, as previously described, the hybridization region comprises aseries of G bases to complement the overhanging C bases at the 3′ end ofa cDNA molecule. The series of G bases may comprise 1 G base, 2 G bases,3 G bases, 4 G bases, 5 G bases or more than 5 G bases. The templatesequence can comprise any sequence to be incorporated into the cDNA. Insome cases, the template region comprises at least 1 (e.g., at least 2,3, 4, 5 or more) tag sequences and/or functional sequences. Switcholigos may comprise deoxyribonucleic acids; ribonucleic acids; modifiednucleic acids including 2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA),inverted dT, 5-Methyl dC, 2′-deoxylnosine, Super T(5-hydroxybutynl-2′-deoxyuridine), Super G (8-aza-7-deazaguanosine),locked nucleic acids (LNAs), unlocked nucleic acids (UNAs, e.g., UNA-A,UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2′ Fluoro bases (e.g., Fluoro C,Fluoro U, Fluoro A, and Fluoro G), or any combination.

In some cases, the length of a switch oligo may be at least about 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197 , 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or250 nucleotides or longer.

In some cases, the length of a switch oligo may be at most about 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197 , 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or250 nucleotides.

Once the contents of the cells are released into their respectivepartitions, the macromolecular components (e.g., macromolecularconstituents of biological particles, such as RNA, DNA, or proteins)contained therein may be further processed within the partitions. Inaccordance with the methods and systems described herein, themacromolecular component contents of individual biological particles canbe provided with unique identifiers such that, upon characterization ofthose macromolecular components they may be attributed as having beenderived from the same biological particle or particles. The ability toattribute characteristics to individual biological particles or groupsof biological particles is provided by the assignment of uniqueidentifiers specifically to an individual biological particle or groupsof biological particles. Unique identifiers, e.g., in the form ofnucleic acid barcodes can be assigned or associated with individualbiological particles or populations of biological particle, in order totag or label the biological particle's macromolecular components (and asa result, its characteristics) with the unique identifiers. These uniqueidentifiers can then be used to attribute the biological particle'scomponents and characteristics to an individual biological particle orgroup of biological particles.

In some aspects, this is performed by co-partitioning the individualbiological particle or groups of biological particles with the uniqueidentifiers, such as described above (with reference to FIG. 2). In someaspects, the unique identifiers are provided in the form of nucleic acidmolecules (e.g., oligonucleotides) that comprise nucleic acid barcodesequences that may be attached to or otherwise associated with thenucleic acid contents of individual biological particle, or to othercomponents of the biological particle, and particularly to fragments ofthose nucleic acids. The nucleic acid molecules are partitioned suchthat as between nucleic acid molecules in a given partition, the nucleicacid barcode sequences contained therein are the same, but as betweendifferent partitions, the nucleic acid molecule can, and do havediffering barcode sequences, or at least represent a large number ofdifferent barcode sequences across all of the partitions in a givenanalysis. In some aspects, only one nucleic acid barcode sequence can beassociated with a given partition, although in some cases, two or moredifferent barcode sequences may be present.

The nucleic acid barcode sequences can include from about 6 to about 20or more nucleotides within the sequence of the nucleic acid molecules(e.g., oligonucleotides). In some cases, the length of a barcodesequence may be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20 nucleotides or longer. In some cases, the length of a barcodesequence may be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 nucleotides or longer. In some cases, the length of abarcode sequence may be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 nucleotides or shorter. These nucleotides may becompletely contiguous, i.e., in a single stretch of adjacentnucleotides, or they may be separated into two or more separatesubsequences that are separated by 1 or more nucleotides. In some cases,separated barcode subsequences can be from about 4 to about 16nucleotides in length. In some cases, the barcode subsequence may beabout 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides orlonger. In some cases, the barcode subsequence may be at least about 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In somecases, the barcode subsequence may be at most about 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

The co-partitioned nucleic acid molecules can also comprise otherfunctional sequences useful in the processing of the nucleic acids fromthe co-partitioned biological particles. These sequences include, e.g.,targeted or random/universal amplification primer sequences foramplifying the genomic DNA from the individual biological particleswithin the partitions while attaching the associated barcode sequences,sequencing primers or primer recognition sites, hybridization or probingsequences, e.g., for identification of presence of the sequences or forpulling down barcoded nucleic acids, or any of a number of otherpotential functional sequences. Other mechanisms of co-partitioningoligonucleotides may also be employed, including, e.g., coalescence oftwo or more droplets, where one droplet contains oligonucleotides, ormicrodispensing of oligonucleotides into partitions, e.g., dropletswithin microfluidic systems.

In an example, microcapsules, such as beads, are provided that eachinclude large numbers of the above described barcoded nucleic acidmolecules (e.g., barcoded oligonucleotides) releasably attached to thebeads, where all of the nucleic acid molecules attached to a particularbead will include the same nucleic acid barcode sequence, but where alarge number of diverse barcode sequences are represented across thepopulation of beads used. In some embodiments, hydrogel beads, e.g.,comprising polyacrylamide polymer matrices, are used as a solid supportand delivery vehicle for the nucleic acid molecules into the partitions,as they are capable of carrying large numbers of nucleic acid molecules,and may be configured to release those nucleic acid molecules uponexposure to a particular stimulus, as described elsewhere herein. Insome cases, the population of beads provides a diverse barcode sequencelibrary that includes at least about 1,000 different barcode sequences,at least about 5,000 different barcode sequences, at least about 10,000different barcode sequences, at least about 50,000 different barcodesequences, at least about 100,000 different barcode sequences, at leastabout 1,000,000 different barcode sequences, at least about 5,000,000different barcode sequences, or at least about 10,000,000 differentbarcode sequences, or more. Additionally, each bead can be provided withlarge numbers of nucleic acid (e.g., oligonucleotide) moleculesattached. In particular, the number of molecules of nucleic acidmolecules including the barcode sequence on an individual bead can be atleast about 1,000 nucleic acid molecules, at least about 5,000 nucleicacid molecules, at least about 10,000 nucleic acid molecules, at leastabout 50,000 nucleic acid molecules, at least about 100,000 nucleic acidmolecules, at least about 500,000 nucleic acids, at least about1,000,000 nucleic acid molecules, at least about 5,000,000 nucleic acidmolecules, at least about 10,000,000 nucleic acid molecules, at leastabout 50,000,000 nucleic acid molecules, at least about 100,000,000nucleic acid molecules, at least about 250,000,000 nucleic acidmolecules and in some cases at least about 1 billion nucleic acidmolecules, or more. Nucleic acid molecules of a given bead can includeidentical (or common) barcode sequences, different barcode sequences, ora combination of both. Nucleic acid molecules of a given bead caninclude multiple sets of nucleic acid molecules. Nucleic acid moleculesof a given set can include identical barcode sequences. The identicalbarcode sequences can be different from barcode sequences of nucleicacid molecules of another set.

Moreover, when the population of beads is partitioned, the resultingpopulation of partitions can also include a diverse barcode library thatincludes at least about 1,000 different barcode sequences, at leastabout 5,000 different barcode sequences, at least about 10,000 differentbarcode sequences, at least at least about 50,000 different barcodesequences, at least about 100,000 different barcode sequences, at leastabout 1,000,000 different barcode sequences, at least about 5,000,000different barcode sequences, or at least about 10,000,000 differentbarcode sequences. Additionally, each partition of the population caninclude at least about 1,000 nucleic acid molecules, at least about5,000 nucleic acid molecules, at least about 10,000 nucleic acidmolecules, at least about 50,000 nucleic acid molecules, at least about100,000 nucleic acid molecules, at least about 500,000 nucleic acids, atleast about 1,000,000 nucleic acid molecules, at least about 5,000,000nucleic acid molecules, at least about 10,000,000 nucleic acidmolecules, at least about 50,000,000 nucleic acid molecules, at leastabout 100,000,000 nucleic acid molecules, at least about 250,000,000nucleic acid molecules and in some cases at least about 1 billionnucleic acid molecules.

In some cases, it may be desirable to incorporate multiple differentbarcodes within a given partition, either attached to a single ormultiple beads within the partition. For example, in some cases, amixed, but known set of barcode sequences may provide greater assuranceof identification in the subsequent processing, e.g., by providing astronger address or attribution of the barcodes to a given partition, asa duplicate or independent confirmation of the output from a givenpartition.

The nucleic acid molecules (e.g., oligonucleotides) are releasable fromthe beads upon the application of a particular stimulus to the beads. Insome cases, the stimulus may be a photo-stimulus, e.g., through cleavageof a photo-labile linkage that releases the nucleic acid molecules. Inother cases, a thermal stimulus may be used, where elevation of thetemperature of the beads environment will result in cleavage of alinkage or other release of the nucleic acid molecules form the beads.In still other cases, a chemical stimulus can be used that cleaves alinkage of the nucleic acid molecules to the beads, or otherwise resultsin release of the nucleic acid molecules from the beads. In one case,such compositions include the polyacrylamide matrices described abovefor encapsulation of biological particles and may be degraded forrelease of the attached nucleic acid molecules through exposure to areducing agent, such as DTT.

In some aspects, provided are systems and methods for controlledpartitioning. Droplet size may be controlled by adjusting certaingeometric features in channel architecture (e.g., microfluidics channelarchitecture). For example, an expansion angle, width, and/or length ofa channel may be adjusted to control droplet size.

FIG. 4 shows an example of a microfluidic channel structure for thecontrolled partitioning of beads into discrete droplets. A channelstructure 400 can include a channel segment 402 communicating at achannel junction 406 (or intersection) with a reservoir 404. Thereservoir 404 can be a chamber. Any reference to “reservoir,” as usedherein, can also refer to a “chamber.” In operation, an aqueous fluid408 that includes suspended beads 412 may be transported along thechannel segment 402 into the junction 406 to meet a second fluid 410that is immiscible with the aqueous fluid 408 in the reservoir 404 tocreate droplets 416, 418 of the aqueous fluid 408 flowing into thereservoir 404. At the juncture 406 where the aqueous fluid 408 and thesecond fluid 410 meet, droplets can form based on factors such as thehydrodynamic forces at the juncture 406, flow rates of the two fluids408, 410, fluid properties, and certain geometric parameters (e.g., w,h₀, α, etc.) of the channel structure 400. A plurality of droplets canbe collected in the reservoir 404 by continuously injecting the aqueousfluid 408 from the channel segment 402 through the juncture 406.

A discrete droplet generated may include a bead (e.g., as in occupieddroplets 416). Alternatively, a discrete droplet generated may includemore than one bead. Alternatively, a discrete droplet generated may notinclude any beads (e.g., as in unoccupied droplet 418). In someinstances, a discrete droplet generated may contain one or morebiological particles, as described elsewhere herein. In some instances,a discrete droplet generated may comprise one or more reagents, asdescribed elsewhere herein.

In some instances, the aqueous fluid 408 can have a substantiallyuniform concentration or frequency of beads 412. The beads 412 can beintroduced into the channel segment 402 from a separate channel (notshown in FIG. 4). The frequency of beads 412 in the channel segment 402may be controlled by controlling the frequency in which the beads 412are introduced into the channel segment 402 and/or the relative flowrates of the fluids in the channel segment 402 and the separate channel.In some instances, the beads can be introduced into the channel segment402 from a plurality of different channels, and the frequency controlledaccordingly.

In some instances, the aqueous fluid 408 in the channel segment 402 cancomprise biological particles (e.g., described with reference to FIGS. 1and 2). In some instances, the aqueous fluid 408 can have asubstantially uniform concentration or frequency of biologicalparticles. As with the beads, the biological particles can be introducedinto the channel segment 402 from a separate channel. The frequency orconcentration of the biological particles in the aqueous fluid 408 inthe channel segment 402 may be controlled by controlling the frequencyin which the biological particles are introduced into the channelsegment 402 and/or the relative flow rates of the fluids in the channelsegment 402 and the separate channel. In some instances, the biologicalparticles can be introduced into the channel segment 402 from aplurality of different channels, and the frequency controlledaccordingly. In some instances, a first separate channel can introducebeads and a second separate channel can introduce biological particlesinto the channel segment 402. The first separate channel introducing thebeads may be upstream or downstream of the second separate channelintroducing the biological particles.

The second fluid 410 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resultingdroplets.

In some instances, the second fluid 410 may not be subjected to and/ordirected to any flow in or out of the reservoir 404. For example, thesecond fluid 410 may be substantially stationary in the reservoir 404.In some instances, the second fluid 410 may be subjected to flow withinthe reservoir 404, but not in or out of the reservoir 404, such as viaapplication of pressure to the reservoir 404 and/or as affected by theincoming flow of the aqueous fluid 408 at the juncture 406.Alternatively, the second fluid 410 may be subjected and/or directed toflow in or out of the reservoir 404. For example, the reservoir 404 canbe a channel directing the second fluid 410 from upstream to downstream,transporting the generated droplets.

The channel structure 400 at or near the juncture 406 may have certaingeometric features that at least partly determine the sizes of thedroplets formed by the channel structure 400. The channel segment 402can have a height, ho and width, w, at or near the juncture 406. By wayof example, the channel segment 402 can comprise a rectangularcross-section that leads to a reservoir 404 having a wider cross-section(such as in width or diameter). Alternatively, the cross-section of thechannel segment 402 can be other shapes, such as a circular shape,trapezoidal shape, polygonal shape, or any other shapes. The top andbottom walls of the reservoir 404 at or near the juncture 406 can beinclined at an expansion angle, α. The expansion angle, α, allows thetongue (portion of the aqueous fluid 408 leaving channel segment 402 atjunction 406 and entering the reservoir 404 before droplet formation) toincrease in depth and facilitate decrease in curvature of theintermediately formed droplet. Droplet size may decrease with increasingexpansion angle. The resulting droplet radius, R_(d), may be predictedby the following equation for the aforementioned geometric parameters ofh₀, w, and α:

$R_{d} \approx {0.44\left( {1 + {2.2\sqrt{\tan \mspace{14mu} \alpha}\frac{w}{h_{0}}}} \right)\frac{h_{0}}{\sqrt{\tan \mspace{14mu} \alpha}}}$

By way of example, for a channel structure with w=21 μm, h=21 μm, andα=3°, the predicted droplet size is 121 μm. In another example, for achannel structure with w=25 μm, h=25 μm, and a=5°, the predicted dropletsize is 123 μm. In another example, for a channel structure with w=28μm, h=28 μm, and α=7°, the predicted droplet size is 124 μm.

In some instances, the expansion angle, α, may be between a range offrom about 0.5° to about 4°, from about 0.1° to about 10°, or from about0° to about 90° . For example, the expansion angle can be at least about0.01°, 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°,4°, 5°, 6°, 7°, 8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°,55°, 60°, 65°, 70°, 75°, 80°, 85°, or higher. In some instances, theexpansion angle can be at most about 89°, 88°, 87°, 86°, 85°, 84°, 83°,82°, 81°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°,20°, 15°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less.In some instances, the width, w, can be between a range of from about100 micrometers ( μm) to about 500 μm. In some instances, the width, w,can be between a range of from about 10 μm to about 200 μm.Alternatively, the width can be less than about 10 μm. Alternatively,the width can be greater than about 500 μm. In some instances, the flowrate of the aqueous fluid 408 entering the junction 406 can be betweenabout 0.04 microliters (μL)/minute (min) and about 40 μL/min. In someinstances, the flow rate of the aqueous fluid 408 entering the junction406 can be between about 0.01 microliters (μL)/minute (min) and about100 μL/min. Alternatively, the flow rate of the aqueous fluid 408entering the junction 406 can be less than about 0.01 μL/min.Alternatively, the flow rate of the aqueous fluid 408 entering thejunction 406 can be greater than about 40 μL/min, such as 45 μL/min, 50μL/min, 55 μL/min, 60 μL/min, 65 μL/min, 70 μL/min, 75 μL/min, 80μL/min, 85 μL/min, 90 μL/min, 95 μL/min, 100 μL/min, 110 μL/min , 120μL/min , 130 μL/min , 140 μL/min , 150 μL/min, or greater. At lower flowrates, such as flow rates of about less than or equal to 10microliters/minute, the droplet radius may not be dependent on the flowrate of the aqueous fluid 408 entering the junction 406.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

The throughput of droplet generation can be increased by increasing thepoints of generation, such as increasing the number of junctions (e.g.,junction 406) between aqueous fluid 408 channel segments (e.g., channelsegment 402) and the reservoir 404. Alternatively, or in addition, thethroughput of droplet generation can be increased by increasing the flowrate of the aqueous fluid 408 in the channel segment 402.

FIG. 5 shows an example of a microfluidic channel structure forincreased droplet generation throughput. A microfluidic channelstructure 500 can comprise a plurality of channel segments 502 and areservoir 504. Each of the plurality of channel segments 502 may be influid communication with the reservoir 504. The channel structure 500can comprise a plurality of channel junctions 506 between the pluralityof channel segments 502 and the reservoir 504. Each channel junction canbe a point of droplet generation. The channel segment 402 from thechannel structure 400 in FIG. 4 and any description to the componentsthereof may correspond to a given channel segment of the plurality ofchannel segments 502 in channel structure 500 and any description to thecorresponding components thereof. The reservoir 404 from the channelstructure 400 and any description to the components thereof maycorrespond to the reservoir 504 from the channel structure 500 and anydescription to the corresponding components thereof.

Each channel segment of the plurality of channel segments 502 maycomprise an aqueous fluid 508 that includes suspended beads 512. Thereservoir 504 may comprise a second fluid 510 that is immiscible withthe aqueous fluid 508. In some instances, the second fluid 510 may notbe subjected to and/or directed to any flow in or out of the reservoir504. For example, the second fluid 510 may be substantially stationaryin the reservoir 504. In some instances, the second fluid 510 may besubjected to flow within the reservoir 504, but not in or out of thereservoir 504, such as via application of pressure to the reservoir 504and/or as affected by the incoming flow of the aqueous fluid 508 at thejunctures. Alternatively, the second fluid 510 may be subjected and/ordirected to flow in or out of the reservoir 504. For example, thereservoir 504 can be a channel directing the second fluid 510 fromupstream to downstream, transporting the generated droplets.

In operation, the aqueous fluid 508 that includes suspended beads 512may be transported along the plurality of channel segments 502 into theplurality of junctions 506 to meet the second fluid 510 in the reservoir504 to create droplets 516, 518. A droplet may form from each channelsegment at each corresponding junction with the reservoir 504. At thejuncture where the aqueous fluid 508 and the second fluid 510 meet,droplets can form based on factors such as the hydrodynamic forces atthe juncture, flow rates of the two fluids 508, 510, fluid properties,and certain geometric parameters (e.g., w, h₀, α, etc.) of the channelstructure 500, as described elsewhere herein. A plurality of dropletscan be collected in the reservoir 504 by continuously injecting theaqueous fluid 508 from the plurality of channel segments 502 through theplurality of junctures 506. Throughput may significantly increase withthe parallel channel configuration of channel structure 500. Forexample, a channel structure having five inlet channel segmentscomprising the aqueous fluid 508 may generate droplets five times asfrequently than a channel structure having one inlet channel segment,provided that the fluid flow rate in the channel segments aresubstantially the same. The fluid flow rate in the different inletchannel segments may or may not be substantially the same. A channelstructure may have as many parallel channel segments as is practical andallowed for the size of the reservoir. For example, the channelstructure may have at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 150, 500, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1000, 1500, 5000 or more parallel or substantiallyparallel channel segments.

The geometric parameters, w, h₀, and α, may or may not be uniform foreach of the channel segments in the plurality of channel segments 502.For example, each channel segment may have the same or different widthsat or near its respective channel junction with the reservoir 504. Forexample, each channel segment may have the same or different height ator near its respective channel junction with the reservoir 504. Inanother example, the reservoir 504 may have the same or differentexpansion angle at the different channel junctions with the plurality ofchannel segments 502. When the geometric parameters are uniform,beneficially, droplet size may also be controlled to be uniform evenwith the increased throughput. In some instances, when it is desirableto have a different distribution of droplet sizes, the geometricparameters for the plurality of channel segments 502 may be variedaccordingly.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

FIG. 6 shows another example of a microfluidic channel structure forincreased droplet generation throughput. A microfluidic channelstructure 600 can comprise a plurality of channel segments 602 arrangedgenerally circularly around the perimeter of a reservoir 604. Each ofthe plurality of channel segments 602 may be in fluid communication withthe reservoir 604. The channel structure 600 can comprise a plurality ofchannel junctions 606 between the plurality of channel segments 602 andthe reservoir 604. Each channel junction can be a point of dropletgeneration. The channel segment 402 from the channel structure 400 inFIG. 2 and any description to the components thereof may correspond to agiven channel segment of the plurality of channel segments 602 inchannel structure 600 and any description to the correspondingcomponents thereof. The reservoir 404 from the channel structure 400 andany description to the components thereof may correspond to thereservoir 604 from the channel structure 600 and any description to thecorresponding components thereof.

Each channel segment of the plurality of channel segments 602 maycomprise an aqueous fluid 608 that includes suspended beads 612. Thereservoir 604 may comprise a second fluid 610 that is immiscible withthe aqueous fluid 608. In some instances, the second fluid 610 may notbe subjected to and/or directed to any flow in or out of the reservoir604. For example, the second fluid 610 may be substantially stationaryin the reservoir 604. In some instances, the second fluid 610 may besubjected to flow within the reservoir 604, but not in or out of thereservoir 604, such as via application of pressure to the reservoir 604and/or as affected by the incoming flow of the aqueous fluid 608 at thejunctures. Alternatively, the second fluid 610 may be subjected and/ordirected to flow in or out of the reservoir 604. For example, thereservoir 604 can be a channel directing the second fluid 610 fromupstream to downstream, transporting the generated droplets.

In operation, the aqueous fluid 608 that includes suspended beads 612may be transported along the plurality of channel segments 602 into theplurality of junctions 606 to meet the second fluid 610 in the reservoir604 to create a plurality of droplets 616. A droplet may form from eachchannel segment at each corresponding junction with the reservoir 604.At the juncture where the aqueous fluid 608 and the second fluid 610meet, droplets can form based on factors such as the hydrodynamic forcesat the juncture, flow rates of the two fluids 608, 610, fluidproperties, and certain geometric parameters (e.g., widths and heightsof the channel segments 602, expansion angle of the reservoir 604, etc.)of the channel structure 600, as described elsewhere herein. A pluralityof droplets can be collected in the reservoir 604 by continuouslyinjecting the aqueous fluid 608 from the plurality of channel segments602 through the plurality of junctures 606. Throughput may significantlyincrease with the substantially parallel channel configuration of thechannel structure 600. A channel structure may have as manysubstantially parallel channel segments as is practical and allowed forby the size of the reservoir. For example, the channel structure mayhave at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,1000, 1500, 5000 or more parallel or substantially parallel channelsegments. The plurality of channel segments may be substantially evenlyspaced apart, for example, around an edge or perimeter of the reservoir.Alternatively, the spacing of the plurality of channel segments may beuneven.

The reservoir 604 may have an expansion angle, α (not shown in FIG. 6)at or near each channel juncture. Each channel segment of the pluralityof channel segments 602 may have a width, w, and a height, h₀, at ornear the channel juncture. The geometric parameters, w, h₀, and α, mayor may not be uniform for each of the channel segments in the pluralityof channel segments 602. For example, each channel segment may have thesame or different widths at or near its respective channel junction withthe reservoir 604. For example, each channel segment may have the sameor different height at or near its respective channel junction with thereservoir 604.

The reservoir 604 may have the same or different expansion angle at thedifferent channel junctions with the plurality of channel segments 602.For example, a circular reservoir (as shown in FIG. 6) may have aconical, dome-like, or hemispherical ceiling (e.g., top wall) to providethe same or substantially same expansion angle for each channel segments602 at or near the plurality of channel junctions 606. When thegeometric parameters are uniform, beneficially, resulting droplet sizemay be controlled to be uniform even with the increased throughput. Insome instances, when it is desirable to have a different distribution ofdroplet sizes, the geometric parameters for the plurality of channelsegments 602 may be varied accordingly.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size. The beads and/orbiological particle injected into the droplets may or may not haveuniform size.

The channel networks, e.g., as described above or elsewhere herein, canbe fluidly coupled to appropriate fluidic components. For example, theinlet channel segments are fluidly coupled to appropriate sources of thematerials they are to deliver to a channel junction. These sources mayinclude any of a variety of different fluidic components, from simplereservoirs defined in or connected to a body structure of a microfluidicdevice, to fluid conduits that deliver fluids from off-device sources,manifolds, fluid flow units (e.g., actuators, pumps, compressors) or thelike. Likewise, the outlet channel segment (e.g., channel segment 208,reservoir 604, etc.) may be fluidly coupled to a receiving vessel orconduit for the partitioned cells for subsequent processing. Again, thismay be a reservoir defined in the body of a microfluidic device, or itmay be a fluidic conduit for delivering the partitioned cells to asubsequent process operation, instrument or component.

The methods and systems described herein may be used to greatly increasethe efficiency of single cell applications and/or other applicationsreceiving droplet-based input. For example, following the sorting ofoccupied cells and/or appropriately-sized cells, subsequent operationsthat can be performed can include generation of amplification products,purification (e.g., via solid phase reversible immobilization (SPRI)),further processing (e.g., shearing, ligation of functional sequences,and subsequent amplification (e.g., via PCR)). These operations mayoccur in bulk (e.g., outside the partition). In the case where apartition is a droplet in an emulsion, the emulsion can be broken, andthe contents of the droplet pooled for additional operations. Additionalreagents that may be co-partitioned along with the barcode bearing beadmay include oligonucleotides to block ribosomal RNA (rRNA) and nucleasesto digest genomic DNA from cells. Alternatively, rRNA removal agents maybe applied during additional processing operations. The configuration ofthe constructs generated by such a method can help minimize (or avoid)sequencing of the poly-T sequence during sequencing and/or sequence the5′ end of a polynucleotide sequence. The amplification products, forexample, first amplification products and/or second amplificationproducts, may be subject to sequencing for sequence analysis. In somecases, amplification may be performed using the Partial HairpinAmplification for Sequencing (PHASE) method.

A variety of applications require the evaluation of the presence andquantification of different biological particle or organism types withina population of biological particles, including, for example, microbiomeanalysis and characterization, environmental testing, food safetytesting, epidemiological analysis, e.g., in tracing contamination or thelike.

Methods for Forming Gel Matrix

The methods and systems described herein may be used to generatediscrete droplets comprising an individual biological particle andpolymer molecules configured to be crosslinked under controlledconditions (e.g., click chemistry). In some cases, the crosslinkedpolymers can be a degradable matrix. In some cases, the crosslinkedpolymers can be a gel. In some cases, the crosslinked polymers can be ahydrogel matrix. In some cases, the crosslinked polymers can enclose abiological sample, e.g., a cell or a nucleic acid. In some cases,additional reagents can permeate into the crosslinked matrix. In somecases, the crosslink(s) between polymer molecules can be cleavable andthe contents enclosed within the matrix can be released after the matrixis cleaved or degraded. In some cases, the crosslinked matrix and/orcontents enclosed therein can be barcoded.

FIG. 7 shows an example of a microfluidic channel structure 700 forco-partitioning biological particles and reagents to generate acrosslinked hydrogel matrix. The channel structure 700 can includechannel segments 701, 702, 704, 706 and 708. Channel segments 701 and702 communicate at a first channel junction 709. Channel segments 701,702, 704, 706, and 708 communicate at a second channel junction 710.

In an example operation, the channel segment 701 may transport anaqueous fluid 712 that includes a plurality of biological particles 714along the channel segment 701 into the second junction 710. As analternative or in addition to, channel segment 701 may transport beads(e.g., gel beads). The beads may comprise barcode molecules.

For example, the channel segment 701 may be connected to a reservoircomprising an aqueous suspension of biological particles 714. Upstreamof, and immediately prior to reaching, the second junction 710, thechannel segment 701 may meet the channel segment 702 at the firstjunction 709. The channel segment 702 may transport a plurality ofreagents 715A (e.g., polymer molecules A) and 715B (e.g., polymermolecules B) suspended in the aqueous fluid 712 along the channelsegment 702 into the first junction 709. For example, the channelsegment 702 may be connected to a reservoir comprising the reagents 715Aand 715B. After the first junction 709, the aqueous fluid 712 in thechannel segment 701 can carry both the biological particles 714 and thereagents 715A and 715B towards the second junction 710. In someinstances, the aqueous fluid 712 in the channel segment 701 can includeone or more reagents, which can be the same or different reagents as thereagents 715A and 715B. A second fluid 716 (e.g., oil) that isimmiscible with the aqueous fluid 712 can be delivered to the secondjunction 710 from each of channel segments 704 and 706. Upon meeting ofthe aqueous fluid 712 from the channel segment 701 and the second fluid716 from each of channel segments 704 and 706 at the second channeljunction 710, the aqueous fluid 712 can be partitioned as discretedroplets 718 in the second fluid 716 and flow away from the secondjunction 710 along channel segment 708. The channel segment 708 maydeliver the discrete droplets 718 to an outlet reservoir fluidly coupledto the channel segment 708, where they may be harvested.

The second fluid 716 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets718.

A discrete droplet generated may include an individual biologicalparticle 714 and/or one or more of reagents 715A and 715B. In someinstances, a discrete droplet generated may include a barcode carryingbead (not shown), such as via other microfluidics structures describedelsewhere herein. In some instances, a discrete droplet may beunoccupied (e.g., no reagents, no biological particles). As shown below,click chemistry can be used to crosslink polymer molecules trapped inthe same discrete droplet to generate hydrogels (e.g., 715A and 715B).In some cases, click chemistry can be used to generate degradablehydrogels (e.g., 715A and 715B).

Gel

As used herein, the term “gel” generally refers to a three-dimensionalpolymeric matrix; a hydrogel is an example of a gel. A gel can have bothliquid and solid characteristics and may exhibit an organized materialstructure. A hydrogel may be a three-dimensional, hydrophilic, polymericmatrix that is configured to absorb/contain water or biological fluids.In some cases, hydrogels can become swollen with water when water is thedispersion medium. See, e.g., Eur. Polym. J., 2015, 65:252-67 and J.Adv. Res, 2015, 6:105-21, each of which is entirely incorporated hereinby reference for all purposes. Hydrogels can take many forms. In somecases, hydrogels can be a water-swollen, cross-linked polymeric networkcreated by crosslinking reactions between monomers. In some cases,hydrogels can be a polymeric material that retaining water within itsmatrix but may not dissolve in water.

Hydrogels can be synthesized in many ways. In some cases, hydrogels canbe synthesized in one-step procedures, e.g., polymerization andconcurrent cross-linking reactions of multifunctional monomers. In somecases, hydrogels can be synthesized in multi-steps procedures, e.g.,polymerization of monomers first, followed by crosslinking reactions byusing orthogonal, reactive groups that can respond to differentconditions to allow stepwise approaches.

Hydrogel products can be classified base on their polymeric compositions(homopolymeric hydrogels, copolymeric hydrogels, or multipolymerhydrogels), types of cross-linking (chemically crosslinked or physicallycrosslinked), physical appearance (matrix, film, or microsphere),network electrical charge (nonionic, ionic, amphoteric or ampholytic, orzwitterionic), and sources (natural (e.g., chitosan) or synthetic (e.g.,polyacrylamide)).

Hydrogels can be synthesized by techniques that can create a crosslinkedpolymer. In some cases, copolymerization/cross-linking free radicalpolymerizations can be used to produce hydrogels by reacting hydrophilicmonomers with multifunctional crosslinking molecules. This can be doneby, for example, linking polymer chains via chemical reaction, usingionizing radiation to generate main-chain free radicals which canrecombine as crosslinking junctions, or physical interactions such asentanglements, electrostatics, and crystallite formation. Types ofpolymerization can include bulk, solution, and suspensionpolymerization.

Suspension polymerization or dispersion polymerization can be employedin water-in-oil or emulsion processes, sometimes called “inversionsuspension.” In some cases, the monomers and initiators can be dispersedin the oil or hydrocarbon phase as a homogenous mixture. In some cases,two types of polymer molecules can be first produced, each having areactive, crosslinking moiety for cross-linking purposes. Then these twotypes of polymer molecules can be enclosed in an emulsion such that thetwo reactive, crosslinking moieties can react and form crosslinksbetween the two types of polymers, thereby completing the synthesis ofthe hydrogel.

In some cases, hydrogels can be synthesized from monomers,polymerization initiators, and crosslinking reagents. After thepolymerization reactions are complete, the hydrogels formed can beseparated from remaining starting materials and unwanted by-products,etc. The length of the polymer formed can be controlled depending on thedesired properties of the hydrogels.

Types of polymerizations employed to synthesize hydrogels can include,but are not limited to, graft polymerization, crosslinkingpolymerization, networks formation of water-soluble polymers, andradiation crosslinking polymerization, etc.

Polymerization can be initiated by initiators or free-radical generatingcompounds, such as, for example, benzoyl peroxide,2,2-azo-isobutyronitrile (AIBN), and ammonium peroxodisulphate, or byusing UV-, gamma- or electron beam-radiation.

In some cases, the hydrogels disclosed herein comprise polymers such aspoly(acrylic acid), poly(vinyl alcohol), poly(vinylpyrrolidone),poly(ethylene glycol), polyacrylamide, some polysaccharides, or anyderivatives thereof. These polymers can be non-toxic, and they can beused in various pharmaceutical and biomedical applications. Thus, insome instances, they may not require their removal from the reactionsystem, thereby eliminating the need for a purification step after theformation of hydrogels.

Polymers can comprise polymer molecules of a particular length or rangeof lengths. Polymer molecules can have a length of at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 1,000,2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000,1,000,000, 2,000,000, 5,000,000, 10,000,000, 20,000,000, 100,000,000,200,000,000, 500,000,000 or 1,000,000,000 backbone atoms or molecules(e.g., carbons). Polymer molecules can have a length of at most 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400,450, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000,500,000, 1,000,000, 2,000,000, 5,000,000, 10,000,000, 20,000,000,100,000,000, 200,000,000, 500,000,000 or 1,000,000,000 backbone atoms ormolecules (e.g., carbons). Polymer molecules can have a length of atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,350, 400, 450, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000,100,000, 200,000, 500,000, 1,000,000, 2,000,000, 5,000,000, 10,000,000,20,000,000, 100,000,000, 200,000,000, 500,000,000 or 1,000,000,000monomer units (e.g., vinyl molecules or acrylamide molecules). Polymermolecules can have a length of at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 1,000, 2,000,5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000,2,000,000, 5,000,000, 10,000,000, 20,000,000, 100,000,000, 200,000,000,500,000,000 or 1,000,000,000 monomer units (e.g., vinyl molecules oracrylamide molecules).

Click Chemistry

As used herein, the term “click chemistry,” generally refers toreactions that are modular, wide in scope, give high yields, generateonly inoffensive byproducts, such as those that can be removed bynonchromatographic methods, and are stereospecific (but not necessarilyenantioselective). See, e.g., Angew. Chem. Int. Ed., 2001,40(11):2004-2021, which is entirely incorporated herein by reference forall purposes. In some cases, click chemistry can describe pairs offunctional groups that can selectively react with each other in mild,aqueous conditions.

An example of click chemistry reaction can be the Huisgen 1,3-dipolarcycloaddition of an azide and an alkynes, i.e., Copper-catalyzedreaction of an azide with an alkyne to form a 5-membered heteroatom ringcalled 1,2,3-triazole. The reaction can also be known as aCu(I)-Catalyzed Azide-Alkyne Cycloaddition (CuAAC), a Cu(I) clickchemistry or a Cu⁺ click chemistry. Catalyst for the click chemistry canbe Cu(I) salts, or Cu(I) salts made in situ by reducing Cu(II) reagentto Cu(I) reagent with a reducing reagent (Pharm Res. 2008, 25(10):2216-2230). Known Cu(II) reagents for the click chemistry can include,but are not limited to, Cu(II)-(TBTA) complex and Cu(II) (THPTA)complex. TBTA, which istris-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, also known astris-(benzyltriazolylmethyl)amine, can be a stabilizing ligand for Cu(I)salts. THPTA, which is tris-(hydroxypropyltriazolylmethyl)amine, can beanother example of stabilizing agent for Cu(I). Other conditions canalso be accomplished to construct the 1,2,3-triazole ring from an azideand an alkyne using copper-free click chemistry, such as by theStrain-promoted Azide-Alkyne Click chemistry reaction (SPAAC, see, e.g.,Chem. Commun., 2011, 47:6257-6259 and Nature, 2015, 519(7544):486-90),each of which is entirely incorporated herein by reference for allpurposes.

The present disclosure also contemplates the use of click chemistryreactions resulting in chemical linkages that are not a 1,2,3-triazole.A range of such click chemistry reactions useful for preparingbiocompatible gels are well-known in the art. See e.g., Madl andHeilshorn, “Bioorthogonal Strategies for Engineering ExtracellularMatrices,” Adv. Funct. Mater. 2018, 28: 1706046, which is herebyincorporated by reference herein.

An example of a click chemistry reaction useful in the compositions andmethods of the present disclosure that is copper-free and does notresult in a 1,2,3-triazole linkage is an Inverse-electron demandDiels-Alder (IED-DA) reaction. (See e.g., Madl and Heilshorn 2018.) Asdescribed elsewhere herein, in the IED-DA click chemistry reaction, thepair of click chemistry functional groups comprises a tetrazine groupand a trans-cyclooctene (TCO) group, or a tetrazine group and anorbonene group. This reaction is copper free and results in a linkagecomprising a dihydropyridazine group rather than a 1,2,3-triazole.

Other specific biorthogonal click chemistry reactions that are useful inthe compositions and methods of the present disclosure, but which resultin a chemical linkage other than a 1,2,3-triazole include a Diels-Alderreaction between a pair of furan and maleimide functional groups, aStaudinger ligation, and nitrile oxide cycloaddition. These clickchemistry reactions and others are well-known in the art and describedin e.g., Madl and Heilshorn 2018.

Accordingly, in some embodiments the copper-free click chemistry usefulin forming crosslinked polymers of the present disclosure can beselected from: (a) strain-promoted azide/dibenzocyclooctyne-amine (DBCO)click chemistry; (b) inverse electron demand Diels-Alder (IED-DA)tetrazine/trans-cyclooctene (TCO) click chemistry; (c) inverse electrondemand Diels-Alder (IED-DA) tetrazine/norbonene click chemistry; (d)Diels-Alder maleimide/furan click-chemistry; (e) Staudinger ligation;and (f) nitrile-oxide/norbonene cycloaddition click chemistry.

For example, a discrete droplet 718 shown in FIG. 7 can be subject tocopper-catalyzed click chemistry conditions shown in FIG. 8. FIG. 8shows an example process of forming hydrogels via click chemistry in anemulsion system. As shown in FIG. 8, emulsion systems 800, 802 and 804can represent different stages through which polymer molecules arecrosslinked to form a hydrogel. Emulsion system 800 can comprise adiscrete droplet 808 (comprising water) immersed in the oil phase 810.Within the discrete droplet 808, two polymer molecules 812 and 814 canbe partitioned together. Polymer molecule 812 can comprise a firstcrosslink precursor comprising a labile bond 816 (e.g., a disulfidebond) and a first click chemistry moiety 818. Polymer molecule 814 cancomprise a second click chemistry moiety 820. In addition, in the oilphase 810, there can be other reagents, such as reagent 822 (shown as acopper (II) reagent) which may be required to facilitate the clickchemistry reaction between the first click chemistry moiety 818 and thesecond click chemistry moiety 820, either by itself or by a derivativethereof. Because the reagent 822 remains outside of the discrete droplet808, generally no click chemistry reaction happens within the discretedroplet 808.

In emulsion system 802, some of the reagent 822 can penetrate into thediscrete droplet 808, via physical or chemical processes. In someinstances, reagent 822 becomes or is otherwise processed to becomereagent 824 (shown as a copper (I) reagent) in the discrete droplet 808.The conversion into reagent 824 can require additional reagents (notshown, e.g., a reducing agent such as sodium ascorbate). In theseembodiments, reagent 824 can be the reagent required to initiate theclick chemistry reaction between the first click chemistry moiety 818and the second click chemistry moiety 820. Once in the proximity of boththe first click chemistry moiety 818 and the second click chemistrymoiety 820, the reagent 824 can initiate a click chemistry reaction,such as a Cu(I)-Catalyzed Azide-Alkyne Cycloaddition (CuAAC), seeemulsion system 804.

As shown in the emulsion system 804 of FIG. 8, in the presence of thereagent 824, a crosslink 826 is formed linking the two polymer molecules812 and 814 together, via the newly formed moiety 828 as a result of theclick chemistry reaction between the first click chemistry moiety 818and the second click chemistry moiety 820. A hydrogel comprising thecrosslinked polymer molecules 812 and 814 can thus be formed. Reagents822 and/or 824 can be removed from the newly formed hydrogel if desired.In some instances, a stimulus (e.g., a chemical, thermal, orphoto-stimulus) is applied to labile bond 816 to release the crosslinks826 and/or degrade the hydrogel.

Copolymer with Click Chemistry Moieties

Scheme 1 below depicts an example synthetic pathway leading to thegeneration of a pair of polymer molecules comprising click chemistrymoieties to be used in a subsequent copolymerization reaction.

Monomer A can react with monomer B to produce polymer C. Monomer B cancomprise a linker L1 between a polymerizable moiety and a clickchemistry moiety CL1. By varying the reaction conditions, the polymer Ccan comprise repeatable units of the formula shown in Scheme 1 in that astretch of n (n is an integer more than 1) repeating regular polymerunits is sandwiched by at least one polymer unit that comprises theclick chemistry moiety CL1, and that there will be a total of m (m is apositive integer) units comprising the click chemistry moiety CL1. Therelative ratio between the repeating regular polymer unit and the unitbearing the click chemistry moiety can be controlled in various ways,include varying the relative amount of the corresponding monomers A andB. The molecular weight and the length of the polymer C can becontrolled by the chain termination conditions. Similarly, monomer A canreact with monomer D to produce polymer E. Monomer D can comprise alinker L2 between a polymerizable moiety and a click chemistry moietyCL2. By varying the reaction conditions, the polymer E can compriserepeatable units of the formula shown in Scheme 1 in that a stretch of n(n is an integer more than 1) repeating regular polymer units issandwiched by at least one polymer unit that comprises the clickchemistry moiety CL2, and that there will be a total of m (m is apositive integer) units comprising the click chemistry moiety CL2. Therelative ratio between the repeating regular polymer unit and thepolymer unit bearing the click chemistry moiety can be controlled invarious ways, include varying the relative amount of the correspondingmonomers A and D. The molecular weight and the length of the polymer Ecan be controlled by the chain termination conditions. The integers nand m are independent in each instance in Scheme 1 in that the polymersC and E can have the same or difference integer n's and m's.

The length and/or the chemical composition of the linkers L1 and L2 canvary, depending on the size of the pore of the hydrogel, the rigidity ofthe linkers, the hydrophilicity of the linkers, etc. Generally, thelinkers L1 and L2 can comprise any chemical groups compatible with thedesired click-chemistry reaction conditions, the desired polymerizationconditions, and/or the desired cell-bead conditions. Accordingly, insome embodiments, the composition of linkers L1 and L2 can comprisechemical groups selected from amine, amide, aryl, imide, carbonate,carbamate, dihydropyridazine, ester, ether, heteroaryl, hydrazone,oxime, phosphodiester, polyethylene glycol (PEG), squarate, thiazole,thiazolidine, thioether, triazole, or any combination thereof. In someembodiments, the composition of linkers L1 and L2 can each comprisealkyl, alkoxy, alkylamino, alkylaminoalkyl, alkoxyalkyl, arylalkyl,arylalkoxy, arylalkylamino, heteroarylalkyl, heteroarylalkoxy,heteroarylalkylamino, or any combinations thereof.

In some embodiments, at least one of the linkers L1 and L2 comprises acopper-chelating chemical group. The presence of a copper-chelatinggroup in the linker L1 or L2 adjacent to the click-chemistry moiety CL1or CL2 can facilitate an accelerated click chemistry reaction due to thechelating group effectively increasing the concentration of the copperion at the site of the reaction. See e.g., Uttamapinant et al., “Fast,Cell-compatible Click Chemistry with Copper-chelating Azides forBiomolecular Labeling,” Angew. Chem. Int. Ed. Eng. 2012 Jun. 11; 51(24):5852-5856, which is hereby incorporated by reference herein.Additionally, the use of a copper-chelating linker with a clickchemistry moiety, such as an azide-picolyl group, can allow for the useof significantly reduced copper concentrations in carrying out a polymercross-linking reaction as described in the methods of the presentdisclosure. In some embodiments, the linkers L1 and L2 comprising acopper-chelating group, such as a picolyl group, can allow a reductionof the copper concentration in the click chemistry reaction by an amountof at least 10%, at least 25%, at least 50%, or at least 75% or more. Inturn, the use of a lower copper concentration can provide for greatlyincreased yields of biological molecules, e.g., reduced RNA degradation,in cell-beads and gel-beads made using the low copper reactions. In someembodiments, the use of copper-chelating groups in linkers can result inan increase in genes detected (e.g., in a cell-bead based geneexpression measurement) by an amount of at least 10%, at least 25%, atleast 50%, or at least 75% or more. The incorporation of the exemplarycopper-chelating group, azide-picolyl, in the click chemistry methods ofthe present disclosure is further described in the Examples.

In some cases, at least one of the linkers L1 and L2 further comprises alabile bond. In some embodiments, linkers L1 and/or L2 can furthercomprise more than one labile bond(s), including bio-orthogonal labilebond(s). Exemplary types of labile bond(s) that can be included in L1and/or L2 can include a chemically labile bond, a thermally labile bond,a photo-labile bond, an enzymatically labile bond, or a combinationthereof. More specific examples of labile bonds that may be part of L1and L2 can include a disulfide bond, an ester linkage, a carbamatelinkage, a vicinal diol linkage, a Diels-Alder linkage, a sulfonelinkage, a silyl ether linkage, a glycosidic linkage, a peptide linkage,or a phosphodiester linkage. The labile bond also can be a bondcleavable by a nucleic acid targeting enzyme, such as restrictionenzyme.

In some embodiments, the linkers L1 and L2 further comprise a disulfidebond. The ability to cleave disulfide bonds with the reductantdithiothreitol (DTT) is well-known in the art. The incorporation oflabile disulfide bonds in linkers L1 and L2 in embodiments of thepresent disclosure are further described in the Examples.

In some cases, however, it is desirable to have DTT present in a reagentmix for a cell-bead based reaction yet still be able to cleave linkersL1 and L2 selectively. Accordingly, in some embodiments, the linkers L1and L2 can further comprise a labile bond that is a carbamate linkage. Acarbamate linkage is not labile in the presence of DTT but can beselectively cleaved by diethylenetriamine (DETA) and heat. In someembodiments, the linkers L1 and L2 can further comprise a labile bondthat is a carbamate linkage, and not comprise a disulfide linkage. Anexemplary labile linker comprising a carbamate group and methods ofcleaving the carbamate and use in the cell-bead based methods of presentdisclosure are further described in the Examples.

In some cases, the linkers L1 and L2 can further comprise a labile bondthat is an enzymatically labile bond. For example, the linkers L1 and L2can include a polypeptide with a sequence that is specifically cleavedby a particular protease. A linker comprising a specific polypeptidesequence with a terminal propargyl moiety can be incorporated into apolymer as shown in FIG. 34A. The propargyl group can then undergo astandard CuAAC click chemistry reaction with an azide modified linker ona second polymer to result in a 1,2,3-triazole crosslinked gel matrix asshown in FIG. 34B. This gel matrix crosslinked by a specific polypeptidesequence can then be selectively degraded enzymatically by exposure to aprotease selective for a peptide linkage in the sequence. As shown inFIG. 34B, a protease, e.g., Type II collagenase, can selectively cleavean 8-mer polypeptide sequence, e.g., GGRMSMPV, at the peptide linkagebetween the M and the S amino acid residues.

Specific proteases are well-known that can selectively cleave a specificpeptide linkage in a polypeptide sequence. It is also contemplated thatthe protease used for degrading polymer crosslinks in a gel does notcleave other peptides/proteins that may be present in a cell-bead basedbiological assay system, such as a polymerase or reverse transcriptase.Exemplary proteases that are highly selective for specific polypeptidesequence and may be used in embodiments related to selectively degradinga hydrogel matrix in the presence of biological particles and othermacromolecular constituents are provided below in Table 1.

TABLE 1 Enzyme Name Cleavage Tag Name Cleavage Sequence/Site HumanRhinovirus 3C (‘PreScission’) LEVLFQ/GP (HRV) 3C cleavage tag (/ = maincleavage site) Protease Enterokinase EKT (Enterokinase) DDDDK/ cleavagetag (/ = main cleavage site) Type II Collagenase — GGRM/SMPV (/ = maincleavage site)

For example, HRV 3C protease is a highly specific protease that cleavesbetween the Q and G residues of the “3C” polypeptide cleavage tagLEVLFQGP and is commercially available “PreScission protease” or “PSP.”Another exemplary highly specific protease is enterokinase. Enterokinaseis an intestinal enzyme normally involved in the protease cleavage oftrypsin that specifically cleaves the peptide linkage after the K of the“EKT” recognition sequence, DDDDK Similarly, as noted above, Type IIcollagenase specifically cleaves the peptide linkages between M and S inthe sequence GGRMSMPV.

In some embodiments, the linker L1 and L2 can comprise a peptide linkageselectively cleavable by a protease. In some embodiments, the linker L1and L2 can comprise a polypeptide comprising a peptide linkageselectively cleavable by a protease, optionally wherein the polypeptidehas a sequence selected from GGRMSMPV, LEVLFQGP, and DDDDK. In someembodiments, the protease is selected from HRV 3C protease,enterokinase, and Type II collagenase.

As described elsewhere herein, a wide range of click chemistry reactionscan be used in generating the polymers for use in the compositions andmethods of the present disclosure. The useful click-chemistry reactionsinclude the well-known copper-catalyzed reactions, such as CuAAC, aswell as copper free click chemistry reactions. Accordingly, the moietiesCL1 and CL2 can comprise any pair of chemical groups that undergo aclick chemistry reaction. In some cases, one of the click chemistrymoieties (CL1 or CL2) comprises an azide while the other comprises analkyne. In some cases, one of the click chemistry moieties (CL1 or CL2)comprises an azide, while the other comprises a dibenzocyclooctyne(DBCO) group.

Both the azide-alkyne CuAAC reaction and the strain-promoted Cu freeazide-DBCO reaction result in a click chemistry linkage comprising a1,2,3-triazole moiety. However, as described elsewhere herein, thepresent disclosure also contemplates gel compositions and methods ofmaking them wherein the linkers and associated click chemistry reactionsfor a linkage that does not comprise a 1,2,3-triazole. Such alternativeclick chemistry reactions and resulting linkages are well-known in theart. See e.g., Madl and Heilshorn, “Bioorthogonal Strategies forEngineering Extracellular Matrices,” Adv. Funct. Mater. 2018, 28:1706046, which is hereby incorporated by reference herein. Accordingly,in some cases, it is contemplated that the click chemistry reaction isan Inverse-electron demand Diels-Alder reaction wherein one of the clickchemistry moieties (CL1 or CL2) comprises a tetrazine group, while theother group comprises a trans-cyclooctene (TCO) group or a norbonenegroup. In both cases, the use of tetrazine with either TCO or norbonene,the click chemistry reaction results in a linkage comprising adihydropyridazine group rather than a 1,2,3-triazole.

Other click chemistry moieties and reactions that do not result in a1,2,3-triazole but can be used in the gel compositions and methods ofmaking them described herein include the furan-maleimide Diels-Alderreaction. Accordingly, in some embodiments, one of the click chemistrymoieties (CL1 or CL2) comprises a furan moiety and the other a maleimidemoiety.

In some cases, the integers n and m are chosen based on the propertiesof the polymers C and E produced. Such properties can include viscosityof the polymers before/after crosslinking, stability, pore sizes of thehydrogels formed, gelation rate, purity, purification procedures,compatibility of the click chemistry moieties with the polymerizationconditions, procedures required to remove the initiators, etc.

Click Chemistry Conditions

As shown in FIG. 8, in some embodiments, a copper (II) species (reagent822) is present in the oil phase outside the discrete droplets whichcomprise polymers to be crosslinked However, in these embodiments,copper (I) species (reagent 824) is the catalyst to enable the clickchemistry reaction inside the discrete droplets. In these instances, thecopper (II) species initially existing in the outside oil phase can beexchanged into the aqueous phase inside the discrete droplets, and thenreduced by a reductant (e.g., sodium ascorbate) in the aqueous phase toproduce the copper (I) species in the aqueous phase inside the discretedroplet.

In some cases, the exchange process can be shown in Scheme 2. To formthe copper (II) species in the oil phase, the first step can be anexchange reaction between copper (II) acetate salt and a fluorinatedcarboxylic acid denoted as Krytox-COOH, which comprises a perfluorinatedalkyl chain to make the compound/complex a stable suspension inperfluorinated oil phase and an acidic carboxylic group to complex thecopper (II). A perfluorinated compound or polymer, such as apoly(perfluoro-propyleneoxide) can be the type of compounds with a nameof. KRYTOX® and produced by DuPont.

Then the Krytox-COO⁻ complexed copper (II) salt can further combine witha perfluorinated surfactant Krytox-PEG-Krytox to form an emulsiondroplet of copper (II) inside the aqueous phase of the droplet. As tothe composition of the droplet thus formed, the surfactantKrytox-PEG-Krytox remain at the interface of the aqueous phase (inside)and the perfluorinated oil phase (outside) with the PEG component of thesurfactant facing the inside aqueous phase and the perfluorinated armspointing toward the outside oil phase. In this way, an oil-phasesuspension of copper (II) species is formed and the oil-phase suspensioncopper (II) species is stable in the oil phase during the time of theclick chemistry reactions. Furthermore, naked copper (II) species and/orthe oil-phase suspension copper (II) species can be removed byfiltration when desired, for example, before or after the clickchemistry reaction is completed.

In some cases, the order of mixing the above reagents (copper (II)acetate, Krytox-COOH, and surfactant Krytox-PEG-Krytox) can beimportant. For example, direct mixing of all three components may notafford the desired suspension of copper (II) species in the oil phase. Astepwise procedure of mixing the copper (II) acetate and Krytox-COOHfirst, followed by the addition of the surfactant, together withstirring/mixing, etc., can produce the desired suspension.

A fluorosurfactant having two fluorophilic tails and one hydrophilichead group (Formula I, hereinafter “tri-block surfactant”) can reducethe coalescence of emulsion droplets and provide stability for emulsionsystems, including, for example, gel bead-in-emulsion systems. Inaddition, a fluorosurfactant having one fluorophilic tail and onehydrophilic head group (Formula II, hereinafter “di-block surfactant” or“di-block copolymer”) may also be used to provide stability for emulsionsystems. Both n and m are integers greater than 1. Krytox-PEG-Krytox isan example of tri-block surfactant.

The second step can be the transport/phase transfer of the dissolvedcopper (II) species into droplets containing polymers to be crosslinked.Various factor can influence the transport/phase transfer, including,but not limited to, the concentration of respective ligands (e.g., THPTAor TBTA) in the oil phase and/or the aqueous phase, other aqueouscomponents in the aqueous phase (e.g., surfactant, magnetic particles,solvents such as water, surfactants such as SYNPERONIC® F-108, the typeand quantity of the reducing agent used (disulfide such asdithiothreitol (DTT) or sodium ascorbate, polymers, and the v/v ratiobetween the oil phase and the aqueous phase during preparation).

The third step can be the reduction of copper (II) species to copper (I)species inside the aqueous phase of the droplet. The reducing reagent,such as, for example, sodium ascorbate, can be chosen based on itschemical properties as a reducing agent and its compatibility with otherparts of the polymers and/or linker groups and/or other reagents thatare present inside the aqueous phase of the droplet. For example, whenthe linker groups (e.g., click chemistry moieties CL1 or CL2) comprise adisulfide bond, using DTT as the copper reducing agent may interferewith the integrity of the polymers or hydrogels because the disulfidebond within the linker groups can be cleaved during the reduction ofcopper (II) species, thereby preventing the desired hydrogels from beinggenerated.

The fourth step can be the click chemistry reaction catalyzed by thecopper (I) species inside the droplet. In some cases, there can be atleast a pair of polymers bearing the two click chemistry moieties 818and 820, respectively, inside the droplet for the click chemistryreaction to occur. In some cases, there can be multiple pairs of suchpolymers. The number of pairs of such polymers inside one droplet can becontrolled during the droplet formation process depicted in FIG. 7.Because crosslinking is involved, there can be cases wherein one polymercrosslinks with more than one other polymers to form the hydrogels.

Factors that can be considered during the click chemistry step mayinclude, but are not limited to, size of the droplets,length/numbers/types/ratio of polymers enclosed in each droplet, ratioof reducing agent to the copper (I)/copper(II) species inside theaqueous phase of the droplet, ratio of copper (I) species to the clickchemistry moieties on the polymers, effect of the ligand (e.g., THPTA orTBTA), time and temperature of the reaction, whether there is externalinfluence (e.g., shaking or vortexing, microwaving, etc.), dissolvedoxygen, and how to separate unwanted reagents/by-products after theclick chemistry reaction is completed.

In some cases, the click chemistry reaction can be run under inert gasconditions. For example, the reaction can be performed under N₂ or Arsuch that oxygen- or air-oxidation of copper (I) species is reduced. Insome cases, the amount of reducing agent added is increased to counterthe side-reaction of this oxygen- or air-oxidation of copper (I)species. In some cases, instead of starting from copper (II) species, acopper (I) species is added in the aqueous fluid as the catalyst for theazide-alkyne cycloaddition. In some cases, when a copper (I) species isthe catalyst initially added in the aqueous fluid for the azide-alkynecycloaddition, an oxygen-free system is provided for the reaction.

In some cases, solvent exchange can be conducted to remove unwantedreagents from the hydrogels formed.

In some cases, further transformations can be performed with thehydrogels by adding additional reagents into or removing some reagentsfrom the hydrogels.

In some cases, a biological sample (e.g., a cell or nucleus or a nucleicacid) is enclosed inside the pores of the hydrogel formed during theclick chemistry reaction. In some cases, the biological sample can bemodified or characterized inside the hydrogel by reacting the biologicalsample with reagents transported into the hydrogel (e.g., through thehydrogel pores).

In some cases, copper nanoparticles can be complexed to a cell which isenclosed inside a hydrogel. In these instances, the copper nanoparticlesare used to catalyze a click chemistry reaction thereby forming thehydrogel. Copper nanoparticles may be used as an alternative to, or inaddition to, copper (II) or copper (I) species. For example, cells maybe complexed with copper nanoparticles prior to partitioning (e.g., intodroplets). Cells comprising the copper nanoparticles are thenpartitioned (e.g., into droplets) with the click chemistry polymersdescribed herein, thereby generating a cross-linked hydrogel. The use ofcell-complexed copper nanoparticles may allow for selective gelation ofa hydrogel, such that a click chemistry reaction is performed only indroplets comprising a copper-complexed cell.

In some cases, during the crosslinking reaction between crosslinkprecursors, the labile bond (e.g., disulfide bond) in the crosslinkprecursors or the crosslink thus formed remains intact (i.e., notbroken). In some cases, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or greater of the labile bond can remain intactduring the crosslinking reaction. In some cases, at least about 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greaterof the labile bond can remain intact during the crosslinking reaction.

In some case, the labile bond (e.g., disulfide bond) in the crosslinkscan be broken by treating with a reagent (e.g., a reducing agent, suchas DTT, TCEP, etc.) such that the biological sample enclosed inside thehydrogel is released and/or the pores of the hydrogel are expanded suchthat one or more reagents capable of reacting with the biological samplecan now gain access to the biological sample.

FIG. 9 shows a schematic of an example hydrogel generated using clickchemistry. A first co-polymer 901 comprises an alkyne moiety 902, and asecond co-polymer 904 comprises an azide moiety 905. Alkyne moiety 902may comprise a degradable linker (e.g., disulfide bond 903).Alternatively, or in addition, azide moiety 905 may comprise adegradable linker. Alkyne moiety 902 may react with azide moiety 905 inthe presence of Cu(I) to generate an interchain linker comprising1,2,3-triazole moiety 906.

Methods for Cell Bead Generation

Methods of the present disclosure may comprise generation of one or morecell beads comprising one or more of the polymers disclosed herein. FIG.10 shows an example method for generating a cell bead. In this example,cells and polymer or gel precursors are mixed with an immiscible fluid(e.g., an oil), thereby generating a plurality of aqueous droplets,including droplet 1001 comprising cell 1002. Droplet 1001 may comprise acharged species, as described herein. Droplet 1001 is subjected toconditions sufficient for polymerization or gelation of the polymer orgel precursors to generate a cell bead 1003 comprising cell 1002.Gelation may comprise any of the gelation mechanisms and polymersdescribed herein, including those utilizing a click chemistry reaction,as described elsewhere herein. In some instances, cell bead 1003 issubjected to treatment conditions sufficient to lyse cell 1002,releasing components of the cell into the cell bead. In otherembodiments, cell 1002 is lysed in droplet 901 prior to polymerizationor gelation of the polymer or gel precursors to generate cell bead 1003.In still other embodiments, cell 1002 is permeabilized before or afterpolymerization or gelation of the polymer or gel precursors. Cell beadsare collected in an aqueous phase to generate a plurality of cell beads1004. Cell beads may be stored for further processing. In some cases,charged species may be attached to the cell beads subsequent topolymerization or gelation of the polymer or gel precursor. Forinstance, polymer or gel precursors may comprise one or more functionalgroups that facilitate the attachment of the charged species subsequentto polymerization or gelation of the polymer or gel precursors. In otherembodiments, the polymer or gel precursors comprise functional groupscomprising the charged species, which are incorporated into the cellbead during polymerization or gelation of the polymer or gel precursors.

In an aspect, the present disclosure provides methods for generating acell bead comprising a charged species. First, a partition may begenerated comprising a cell from a plurality of cells, a polymeric orgel precursor, and a charged species. Next, the partition may besubjected to conditions sufficient to react the polymeric or gelprecursor to generate a polymer or gel network comprising the cell or aderivative thereof and the charged species, thereby generating a cellbead. The partition may be subjected to conditions sufficient topolymerize or gel the polymeric or gel precursors. Conditions sufficientto polymerize or gel polymeric or gel precursors are described elsewhereherein. In some embodiments, the cell is lysed to release components ofthe cell into the cell bead. The cell may be lysed prior topolymerization or gelling of the polymeric or gel precursors,concurrently with polymerization or gelling of the polymeric or gelprecursors, or subsequent to polymerization or gelling of the polymericor gel precursors. In other embodiments, the cell in the cell bead isnot lysed, but is permeabilized to allow access to components within thenucleus.

In another aspect, the present disclosure provides methods forgenerating a cell bead comprising a charged species. First, a partitionmay be generated comprising a nucleus isolated from a cell, a polymericor gel precursor, and a charged species. Next, the partition may besubjected to conditions sufficient to react the polymeric or gelprecursors to generate a polymer or gel network comprising the nucleusand the charged species, thereby generating a cell bead. The partitionmay be subjected to conditions sufficient to polymerize or gel thepolymeric or gel precursors. Conditions sufficient to polymerize or gelpolymeric or gel precursors are described elsewhere herein. For example,a copper catalyst may be used to catalyze a click chemistry reaction,thereby generating a hydrogel. In some embodiments, the nucleus is lysedto release components of the nucleus into the cell bead. The nucleus maybe lysed prior to polymerizing or gelling the polymeric or gelprecursors, concurrently with polymerizing or gelling the polymeric orgel precursors, or subsequent to polymerizing or gelling the polymericor gel precursors. In other embodiments, the nucleus in the cell bead isnot lysed, but is permeabilized to allow access to nuclear componentswithin the nucleus.

A charged species may be a positively charged species. A positivelycharged species may be a reagent comprising a positive charge. Apositively charged species may comprise trimethylammonium. A positivelycharged species may be (3-Acrylamidopropyl)-trimethylammonium. A chargedspecies may be a negatively charged species. A negatively chargedspecies may comprise phosphate. A charged species may be attached to thepolymer or gel network. A charged species may be incorporated into apolymer or gel network during polymerization. A cell bead may compriseone or more chemical cross-linkers. A chemical cross-linker may be adisulfide bond. A charged species may be attached to one or morechemical cross-linkers. A derivative of a cell may be a component from acell (e.g., DNA, RNA, protein, etc.). A method of generating a cell beadmay comprise lysing a cell within a partition (e.g., a droplet) torelease a component from the cell. A component may be a nucleic acid. Anucleic acid may be DNA (e.g., genomic DNA) or RNA (e.g., mRNA, siRNA).A component may be a protein. A component may be a negatively chargedcomponent, for example, DNA, RNA, or miRNA. A component may be apositively charged component, for example, a protein. A component from acell may interact with a charged species. A component from a cell may benon-covalently attached to a charged species.

In some embodiments, a negatively charged component from or derived froma cell (e.g., DNA) interacts with a positively charged species (e.g.,((3-Acrylamidopropyl)-trimethylammonium) of the cell bead (e.g., apositively charged functional group of the cell bead polymers) via ionicinteractions. In other embodiments, a positively charged component fromor derived from a cell (e.g., a protein) interacts with a negativelycharged species (e.g., phosphate) of the cell bead (e.g., a negativelycharged functional group of the cell bead polymers) via ionicinteractions. In still other embodiments, a negatively charged componentfrom or derived from a cell (e.g., DNA) interacts with a positivelycharged species (e.g., ((3-Acrylamidopropyl)-trimethylammonium) of thecell bead (e.g., a positively charged functional group of the cell beadpolymers) and a positively charged component from or derived from a cell(e.g., a protein) interacts with a negatively charged species (e.g.,phosphate) of the cell bead (e.g., a negatively charged functional groupof the cell bead polymers). Thus, one or more components from a cell maybe capable of being retained within the cell bead, for example, due toelectrostatic interactions with a charged species of a cell bead. Acomponent from a cell may be capable of being retained within the cellbead for about 1 hour, about 2 hours, about 3 hours, about 4 hours,about 5 hours, about 6 hours, about 12 hours, about 24 hours, about 48hours, about 72 hours, or more. A component from a cell may be capableof being retained within the cell bead for at least 1 hour, at least 2hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6hours, at least 12 hours, at least 24 hours, at least 48 hours, at least72 hours, or more. A component from a cell may be capable of beingretained within the cell bead for at most 1 hour, at most 2 hours, atmost 3 hours, at most 4 hours, at most 5 hours, at most 6 hours, at most12 hours, at most 24 hours, at most 48 hours, or at most 72 hours.

In an aspect, the present disclosure provides methods for generating acell bead comprising an electrically charged polymer or gel network(e.g., a cell bead comprising a charged species). First, a partition maybe generated comprising a cell from a plurality of cells and a polymericor gel precursor. Next, the partition may be subjected to conditionssufficient to react said electrically charged polymeric or gel precursorto generate an electrically charged polymer or gel network comprisingthe cell or a derivative thereof, thereby providing the cell beadcomprising the charged species. The reaction may be such that the netcharge on the polymer or gel precursor is changed, thereby generating anelectrically charged polymer or gel network. The reaction may be suchthat the net charge on the polymer or gel network is changed, therebygenerating an electrically charged polymer or gel network.

The polymer or gel precursor may be positively charged. The polymer orgel precursor may comprise chitosan. The polymer or gel precursor maycomprise polyethyleneimine (PEI). The polymer or gel precursor may benegatively charged. The polymer or gel precursor may comprise alginate.A derivative of a cell may be a component from a cell (e.g., DNA, RNA,protein, etc.). A method of generating a cell bead may comprise lysing acell within a partition (e.g., a droplet) to release a component fromthe cell. A component may be a nucleic acid. A nucleic acid may be DNA(e.g., genomic DNA) or RNA (e.g., mRNA, siRNA). A component may be aprotein. A component may be a negatively charged component, for example,DNA, RNA, or miRNA. A component may be a positively charged component,for example, a protein. A component from a cell may interact with theelectrically charged polymer or gel network. A component from a cell maybe non-covalently attached to the polymer or gel network of a cell beadcomprising a charged species. In some embodiments, a negatively chargedcomponent from or derived from a cell (e.g., DNA) interacts with apositively charged species of the cell bead (e.g., a positive chargedpolymer or gel network) via ionic interactions. In other embodiments, apositively charged component from or derived from a cell (e.g., aprotein) interacts with a negatively charged species of the cell bead(e.g., a negatively charged polymer or gel network) via ionicinteractions. In still other embodiments, a negatively charged componentfrom or derived from a cell (e.g., DNA) interacts with a positivelycharged species of the cell bead (e.g., a positive charged polymer orgel network) and a positively charged component from or derived from acell (e.g., a protein) interacts with a negatively charged species ofthe cell bead (e.g., a negatively charged polymer or gel network). Thus,one or more components from a cell may be capable of being retainedwithin the cell bead, for example, due to electrostatic interactionswith a charged species of a cell bead. A component from a cell may becapable of being retained within the cell bead, for example, due tointeractions with an electrically charged polymer or gel network. Acomponent from a cell may be capable of being retained within the cellbead for about 1 hour, about 2 hours, about 3 hours, about 4 hours,about 5 hours, about 6 hours, about 12 hours, about 24 hours, about 48hours, about 72 hours, or more. A component from a cell may be capableof being retained within the cell bead for at least 1 hour, at least 2hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6hours, at least 12 hours, at least 24 hours, at least 48 hours, at least72 hours, or more. A component from a cell may be capable of beingretained within the cell bead for at most 1 hour, at most 2 hours, atmost 3 hours, at most 4 hours, at most 5 hours, at most 6 hours, at most12 hours, at most 24 hours, at most 48 hours, or at most 72 hours.

In an aspect, the present disclosure provides methods for generating acell bead comprising a charged species. First, a partition may begenerated comprising a cell from a plurality of cells and a polymeric orgel precursor. Next, the partition may be subjected to conditionssufficient to react the polymeric or gel precursor to generate a polymeror gel network comprising the cell or a derivative thereof. Next, acharged species may be coupled to the polymer or gel network, therebyproviding the cell bead comprising the charged species. The partitionmay be subjected to conditions sufficient to polymerize or gel thepolymeric or gel precursors. Conditions sufficient to polymerize or gelpolymeric or gel precursors are described elsewhere herein. For example,a copper catalyst may be used to catalyze a click chemistry reaction,thereby generating a hydrogel. In some cases, the cell is lysed torelease cellular components. The cell may be lysed prior to polymerizingor gelling the polymeric or gel precursors, concurrently withpolymerizing or gelling the polymeric or gel precursors, or subsequentto polymerizing or gelling the polymeric or gel precursors.

A polymer or gel network can be a degradable polymer or gel network, asdescribed herein, such that a cell bead is a degradable cell bead. Anynumber of cell beads may be generated by generating a plurality ofpartitions. In some cases, about 1, about 2, about 3, about 4, about 5,about 10, about 50, about 100, about 500, about 1000, about 5000, about10000, about 20000, about 50000, about 100000, or more cell beads aregenerated, thereby generating a plurality of cell beads. A cell bead maybe partitioned together with a barcode bead (e.g., a gel bead) foranalysis of a cell or components thereof. Compositions for CellularAnalysis

Disclosed herein are compositions comprising a cell bead comprising apolymerized or cross-linked polymer network comprising a cell or a lysedcell generated from the cell, wherein the polymerized or cross-linkedpolymer network is electrically charged. The cell bead may comprise acomponent from a cell. A component may be a nucleic acid. A nucleic acidmay be DNA (e.g., genomic DNA) or RNA (e.g., mRNA, miRNA). A componentmay be a protein. The polymer network may be positively charged. Thepolymer network may comprise chitosan. The polymer network may comprisePEI. The polymer network may be negatively charged. The polymer networkmay comprise alginate. A component from a cell may be capable of beingretained within the cell bead for about 1 hour, about 2 hours, about 3hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours,about 24 hours, about 48 hours, about 72 hours, or more. A componentfrom a cell may be capable of being retained within the cell bead for atleast 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, atleast 5 hours, at least 6 hours, at least 12 hours, at least 24 hours,at least 48 hours, at least 72 hours, or more. A component from a cellmay be capable of being retained within the cell bead for at most 1hour, at most 2 hours, at most 3 hours, at most 4 hours, at most 5hours, at most 6 hours, at most 12 hours, at most 24 hours, at most 48hours, or at most 72 hours.

Also disclosed herein are compositions comprising a cell bead comprisinga polymerized or cross-linked polymer network comprising a cell or alysed cell generated from the cell and a charged species. The cell beadmay comprise a component from the cell. A component may be a nucleicacid. A nucleic acid may be DNA (e.g., genomic DNA) or RNA (e.g., mRNA,miRNA). A component may be a protein. The charged species may bepositively charged. The charged species may comprise trimethylammonium .The charged species may be (2-Aminoethyl)trimethylammonium . The chargedspecies may be (3-Acrylamidopropyl)trimethylammonium. The chargedspecies may be negatively charged. The charged species may comprisephosphate. A component from a cell may be capable of being retainedwithin the cell bead for about 1 hour, about 2 hours, about 3 hours,about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 24hours, about 48 hours, about 72 hours, or more. A component from a cellmay be capable of being retained within the cell bead for at least 1hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5hours, at least 6 hours, at least 12 hours, at least 24 hours, at least48 hours, at least 72 hours, or more. A component from a cell may becapable of being retained within the cell bead for at most 1 hour, atmost 2 hours, at most 3 hours, at most 4 hours, at most 5 hours, at most6 hours, at most 12 hours, at most 24 hours, at most 48 hours, or atmost 72 hours.

FIG. 11A shows example cell beads of the present disclosure comprisingpositively charged species attached to a polymer or gel network. Cellbeads 1101 comprise positively charged species 1103 attached to thepolymer or gel network. Cell Beads 1101 comprising positively chargedspecies 1103 can be generated using any of the methods disclosed herein,including polymerization or gelling of electrically charged polymeric orgel precursors (e.g., gel precursors comprising charged functionalgroups). Cell beads 1101 also comprise a negatively charged cellcomponent 1102 (e.g., nucleic acid, such as DNA) from a single cell.Positively charged species 1103 interacts with the negatively chargedcell component 1102, thereby retaining the negatively charged cellcomponent 1102 in the cell beads 1101. Cell beads 1101 are stored withlittle to no diffusion of the negatively charged cell component 1102 outof the cell beads over time.

FIG. 11B shows an example cell bead of the present disclosure comprisingnegatively charged species attached to a polymer or gel network. Cellbeads 1111 comprise negatively charged species 1113 attached to thepolymer or gel network. Cell Beads 1111 comprising negatively chargedspecies 1113 can be generated using any of the methods disclosed herein,including polymerization or gelling of electrically charged polymeric orgel precursors (e.g., gel precursors comprising charged functionalgroups). Cell beads 1111 also comprise a positively charged cellcomponent 1112 (e.g., a protein or polypeptide comprising a region ofnet positive charge) from a single cell. Negatively charged species 1113interact with the positively charged cell component 1112, therebyretaining the positively charged cell component 1112 in the cell beads1111. Cell beads 1111 are stored with little to no diffusion of thepositively charged cell component 1112 out of the cell beads.

FIG. 12A shows example cell beads of the present disclosure comprisingpositively charged species attached to releasable chemicalcross-linkers. Cell beads 1201 comprise positively charged species 1203attached to releasable chemical cross-linkers 1204 (e.g., disulfidebonds). Cell beads 1201 comprising positively charged species 1203attached to releasable chemical cross-linkers 1204 can be generatedusing any of the methods disclosed herein, including polymerization orgelling of gel precursors comprising charged functional groups attachedto releasable chemical cross-linkers. In other embodiments, cell beads1201 comprising positively charged species 1203 attached to releasablechemical cross-linkers 1204 are generated by polymerization or gellingof gel precursors, followed by functionalization of the polymerized orgelled cell bead with charged functional groups using a cross-linkingagent. Cell beads 1201 also comprise a negatively charged cell component1202 (e.g., nucleic acid, such as DNA) from a single cell. Positivelycharged species 1203 interacts with the negatively charged cellcomponent 1202, thereby retaining the negatively charged cell component1202 in the cell beads 1201. Cell beads 1201 are stored with little tono diffusion of the negatively charged cell component 1202 out of thecell beads over time.

FIG. 12B shows an example of a cell bead of the present disclosurecomprising negatively charged species attached to releasable chemicalcross-linkers. Cell beads 1211 comprise negatively charged species 1213attached to releasable chemical cross-linkers 1204 (e.g., disulfidebonds). Cell beads 1211 comprising negatively charged species 1213attached to releasable chemical cross-linkers 1214 can be generatedusing any of the methods disclosed herein, including polymerization orgelling of gel precursors comprising charged functional groups attachedto releasable chemical cross-linkers. In other embodiments, cell beads1211 comprising negatively charged species 1213 attached to releasablechemical cross-linkers 1214 are generated by polymerization or gellingof gel precursors, followed by functionalization of the polymerized orgelled cell bead with charged functional groups using a cross-linkingagent. Cell beads 1211 also comprise a positively charged cell component1212 (e.g., a protein or polypeptide comprising a region of net positivecharge) from a single cell. Negatively charged species 1213 interactswith a positively charged cell component 1212, thereby retaining thepositively charged cell component 1212 in the cell beads 1211. Cellbeads 1211 are stored with little to no diffusion of positively chargedcell component 1212 out of the cell beads over time.

The charged cell beads and charged hydrogels of the present disclosure(e.g., FIG. 11A-B, FIG. 12A-B) can also comprise any of the crosslinkedpolymers (e.g., click chemistry polymers, such as in FIG. 8) asdisclosed elsewhere herein.

Cell Beads

In an aspect, the present disclosure provides methods and systems forthe generation of cell beads, which may be useful in processingdifferent components from single cells. Cell beads may be generated bymethods as described herein, for example by polymerization of molecularprecursors (e.g., polymer or gel precursors) in a partition comprising acell or constituents from a cell. Cell beads can comprise one or moredifferent types of components from a cell, including, for example, DNA(e.g., gDNA, chromatin, etc.), RNA (e.g., mRNA, miRNA), proteins, and/ormetabolites. Components may be comprised in and/or attached to cellbeads. Cell beads can be generated by encapsulating a cell in a polymeror gel matrix and lysing the cell in the gel or polymer matrix, lysingthe cell while it is being encapsulated in the polymer or gel matrix, orlysing the cell so that its constituents are encapsulated in the polymeror gel matrix. The polymer or gel matrix may comprise one or morecharged species configured to interact with a component from a cell(e.g., DNA, RNA, proteins, etc.).

The partition used in generating a cell bead may comprise one or morereagents for conducting one or more reactions. Species may include, forexample, reagents for a nucleic acid amplification or extension reaction(e.g., primers, polymerases, nucleotides, co-factors (e.g., ionicco-factors), buffers, etc.) including those described herein, reagentsfor enzymatic reactions (e.g., enzymes, co-factors, substrates, buffers,etc.), reagents for nucleic acid modification reactions, such aspolymerization, ligation, or digestion, and/or reagents for templatepreparation.

Reagents may comprise reagents for minimizing damage of nucleic acidsresulting from a click chemistry reaction. For example, a radicalscavenger may be added to a partition, thereby reducing the risk ofdamage to nucleic acids caused by free radicals generated during a clickchemistry reaction. In some cases, the radical scavenger comprisesdimethyl sulfoxide (DMSO). DMSO may be added to a partition used ingenerating a cell bead at a sufficient concentration for preventingnucleic acid damage. In some embodiments, DMSO is added to a partitionat an amount of at least about 1%, about 2%, about 3%, about 4%, about5%, about 6%, about 7%, about 8%, about 9%, about 10%, or greater.

One or more reagents within a partition may be attached to precursors(e.g., polymer or gel precursors). Reagents may be covalently attachedto precursors. Reagents may be reversibly or irreversible attached toprecursors. Reagents may be attached to precursors via an acryditemoiety.

In some cases, oligonucleotides may be attached to the precursors.Oligonucleotides attached to precursors may be useful in, for example,capturing RNA and/or performing a reverse transcription reaction.Oligonucleotides may comprise a poly-T sequence or a poly-U sequence(e.g., may be a poly-T primer). In some embodiments, a poly-T sequenceis used to hybridize to a poly-A sequence, for example, from mRNA of acell. In some embodiments, a poly-U sequence is used to hybridize to apoly-A sequence, for example, from mRNA of a cell.

In some cases, an oligonucleotide, such as a poly-T sequence, can beattached to a precursor (e.g., polymer) via an irreversible clickchemistry reaction. In some embodiments, this click chemistry attachmentof an oligonucleotide can be carried out during the crosslinking of thepolymers that results in a gel matrix. For example, as depicted in FIG.35, a propargylated poly-T oligonucleotide introduced into an emulsiondroplet together with the polymers modified with azide and alkyne clickchemistry groups is attached via CuAAC click chemistry resulting in a1,2,3-triazole linkage with some of the azide-modified linker sites ofthe azide-modified polymer. The other sites form crosslinks with thealkyne-modified polymers resulting in a gel matrix comprising covalentlyattached poly-T reagents capable of capturing polyadenylated RNAtranscripts.

A partition used in generating a cell bead may comprise one or moreparticles (e.g., magnetic particles). One or more reagents within apartition may be attached to the particle. Reagents may be covalentlyattached to the particle. Reagents may be reversibly or irreversiblyattached to the particle. Regents may be attached to the particle via anacrydite moiety. In some cases, oligonucleotides may be attached to theparticle. Oligonucleotides attached to the particle may be useful in,for example, capturing RNA and performing a reverse transcriptionreaction. In some embodiments, the particles (which are optionallymagnetic particles) comprise oligonucleotides attached thereto thatcomprise a poly-T sequence capable of hybridizing to a poly-A sequence,for example, from mRNA of a cell.

A cell within a partition may be lysed as described herein, therebyreleasing constituents from the cell into the partition. Constituentsmay include multiple types of cellular components, including proteins,metabolites, and/or nucleic acid molecules (e.g., DNA, RNA (e.g.messenger RNA), etc.). Alternatively, or in addition, a cell within apartition may by permeabilized. Permeabilization may allow for transferof certain reagents, species, constituents, etc. into and/or out of acell with or without complete cellular lysis. In some embodiments, thecell is lysed or permeabilized prior to the polymerization or gelling ofthe cell bead. In other embodiments, the cell is lysed or permeabilizedconcurrent with the polymerization or gelling of the cell bead. In someembodiments, the cell is lysed or permeabilized subsequent to thepolymerization or gelling of the cell bead. In still other embodiments,the cell is not lysed or permeabilized while in the cell bead.

Reagents can be included within a partition, including reagents attachedto precursors, particles, etc., and may be used to perform one or morereactions on the cell or constituents from or derived from a cell. Areaction may be, for example, amplification, reverse transcription, ordeamination reaction. In some embodiments, the one or more reactions areperformed prior to the polymerization or gelling of the cell bead. Insome embodiments, the one or more reactions are performed concurrentwith the polymerization or gelling of the cell bead. In someembodiments, the one or more reactions are performed subsequent to thepolymerization or gelling of the cell bead. In some cases,oligonucleotides (e.g., primers) are used to perform a reversetranscription reaction on messenger RNA from a cell, thereby generatingcomplementary DNA (cDNA). Reverse transcription may comprise theaddition of additional nucleotides, e.g., a polynucleotide such aspolyC, to the cDNA. In some cases, template switching may be performedto further extend the cDNA. Template switching may append one or moreadditional sequences to the cDNA. Additional sequences may, in somecases, be used to facilitate nucleic acid extension/amplification and/orbarcoding, as described herein. cDNA may be attached to precursorsand/or particles. In some cases, oligonucleotides are used to capturemessenger RNA from a cell, (e.g., via hybridization) prior to generationof a cell bead.

FIG. 13 illustrates an example method for generating cDNA from cellularmRNA and attaching the cDNA to a polymeric precursor. A partition 1300(e.g., an aqueous droplet in an emulsion) may comprise a cell 1301 andan oligonucleotide 1302 attached to a polymeric precursor 1310. In someembodiments, the oligonucleotide 1302 comprises a poly-T sequence,random N-mer, targeted capture/primer sequence, and/or any otheradditional sequences, such as the functional sequences describedelsewhere herein. In some instances, the partition 1300 furthercomprises one or more reagents, such as reagents for performing one ormore reactions on one or more components of the cell (e.g., a reversetranscriptase enzyme, buffer, cofactors, etc.) or reagents forpolymerizing or gelling the polymeric precursor 1310. The partition 1300can also optionally comprise a template switching oligonucleotide (notshown). Cell 1301 is lysed or permeabilized, thereby releasing orotherwise allowing access to multiple types of cellular constituentsincluding messenger RNA (mRNA) 1303 and genomic DNA (gDNA) 1305.Oligonucleotide 1302 may be used to perform reverse transcription (RT)of the mRNA, thereby generating complementary DNA (cDNA) 1304 attachedto polymeric precursor 1310. In some instances, a template switchingreaction can be performed using the template switching oligonucleotideto, e.g., append additional sequences to the cDNA. The polymericprecursor 1310 comprising the cDNA can then be polymerized or gelled togenerate a cell bead comprising cDNA 1304 and gDNA 1305. In someembodiments, the polymeric precursor 1310 is polymerized or gelled toform a cell bead comprising mRNA 1303 (which may be hybridized tooligonucleotide 1302) and gDNA 1305 and cDNA 1304 is generated in thecell bead. In some embodiments, the oligonucleotide 1302 is releasablyattached to the gel precursor 1310 via a labile bond.

FIG. 14 illustrates an example method for capturing cellular mRNA orgenerating cDNA using an oligonucleotide attached to a magneticparticle. A partition 1400 (e.g., an aqueous droplet in an emulsion) maycomprise a cell 1401, an oligonucleotide 1402 attached to a particle1403 (e.g., a bead or magnetic particle), and a polymeric precursor1410. In some embodiments, the oligonucleotide 1402 comprises a poly-Tsequence, random N-mer, targeted capture/primer sequence, and/or anyother additional sequences, such as the functional sequences describedelsewhere herein. In some instances, the partition 1400 furthercomprises one or more reagents, such as reagents for performing one ormore reactions on one or more components of the cell (e.g., a reversetranscriptase enzyme, buffer, cofactors, etc.) or reagents forpolymerizing or gelling the polymeric precursor 1410. Cell 1401 may belysed or permeabilized, thereby releasing or otherwise allowing accessto multiple types of cellular constituents including mRNA 1404 andgenomic DNA (gDNA) 1405. The mRNA 1404 is then subjected to conditionssuch that it hybridizes to the oligonucleotide 1402 (e.g., via a poly-Tsequence), thereby capturing the mRNA. In some embodiments, thehybridized mRNA 1404 is converted into cDNA. The polymeric precursor1410 can then be polymerized or gelled to generate a cell beadcomprising the particle attached mRNA 1404 (or cDNA) and gDNA 1405.Thus, the captured mRNA 1404 (e.g., hybridized to the oligonucleotide1402 coupled to the particle 1403) is incorporated into the cell bead.In some instances, the captured mRNA 1404 or cDNA can be purified awayfrom the partition 1400 and processed separately.

In some embodiments, a partition is subjected to conditions sufficientto generate a cell bead comprising one or more reagents. For example, apartition droplet comprising polymer precursors attached to reagents(e.g., primers, nucleic acid molecules, etc.) may be polymerized orgelled such that the reagents are attached to the polymer or gel matrix(i.e., attached to a cell bead). In some instances, the reagents arereleasably attached to the gel precursor via a labile bond (e.g., achemically labile bond, thermally labile bond, or photo-labile bond).Reagents may be covalently attached to a cell bead. Reagents may bereversible or irreversibly attached to a cell bead. Reagents may beattached to the surface of a gel bead. Reagents may be attached to theinside of a cell bead. In some cases, mRNA is attached to a cell bead.For example, polymer precursors attached to mRNA from a cell may bepolymerized or gelled to generate a cell bead such that the mRNA isattached to the cell bead. In some cases, cDNA is attached to a cellbead. For example, polymer precursors attached to cDNA derived from acell may be polymerized to generate a cell bead such that the cDNA isattached to the cell bead. In some cases, one or more oligonucleotidesare attached to a cell bead. For example, polymer precursors attached tothe oligonucleotides may be polymerized or gelled to generate a cellbead such that the oligonucleotides are attached to the cell bead.

FIG. 15 illustrates an example of generating cell beads comprisingreagents attached to a polymer matrix. A partition 1500 comprisingpolymer precursors 1510 attached to nucleic acid molecules 1502 and 1503(e.g., mRNA, cDNA, primers, etc.) may be subjected to conditionssufficient to polymerize the polymer precursors, thereby generating acell bead 1511 comprising nucleic acid molecules 1502 and 1503 attachedto the polymer matrix 1530. In some instances, a partition 1550comprises a first type of polymer precursor 1510 and a second type ofpolymer precursor 1520 and a cell bead 1511 is generated comprising aco-polymer 1530 of polymer precursors 1510 and 1520. In some instances,a nucleic acid molecule is attached to the first and/or second polymerprecursors. For instance, a nucleic acid molecule 1502 can be attachedto the first type of polymer precursor 1510 and a nucleic acid molecule1503 can be attached to the second type of polymer precursor 1520 and acell bead 1511 is generated comprising nucleic acid molecules 1502 and1503 attached to the polymer matrix 1540. In some instances, nucleicacid molecule 1502 and nucleic acid molecule 1503 are identical. In someinstances, nucleic acid molecule 1502 and nucleic acid molecule 1503 arethe same type of molecule (e.g., mRNA or cDNA), but may containdifferent sequences. In other instances, nucleic acid molecule 1502 andnucleic acid molecule 1503 are different.

Attaching macromolecular constituents (e.g., nucleic acid molecules,protein, etc.) to a cell bead or a particle within a cell bead may beuseful in preparing the species for further processing. For example,nucleic acid molecules attached to a cell bead or particle may beprocessed while remaining attached to the cell bead or particle.Following processing, a nucleic acid may be released (e.g., releasedinto a partition) from a cell bead and/or particle for analysis. In somecases, it may be useful to attach one type of cellular component orderivative thereof (e.g., mRNA, cDNA) to a cell bead or a particlewithin a cell bead, while encapsulating but not attaching another typeof cellular component (e.g., genomic DNA). This may be useful in, forexample, facilitating separate processing of multiple types ofcomponents. For example, following cell bead formation, cell beads maybe transferred to an aqueous solution and subjected to additionalprocessing as described herein. For example, cell beads may besubjected, in bulk, to reverse transcription to generate cDNA fromcaptured mRNA (e.g., hybridized to an oligonucleotide attached to thecell bead matrix or a particle, such as a magnetic particle).

Partitioning Cell Beads

Cell beads may be partitioned together with nucleic acid barcodemolecules and the nucleic acid molecules of or derived from the cellbead (e.g., mRNA, cDNA, gDNA, etc.,) can be barcoded as describedelsewhere herein. In some embodiments, cell beads are co-partitionedwith barcode carrying beads (e.g., gel beads) and the nucleic acidmolecules of or derived from the cell bead are barcoded as describedelsewhere herein. An overview of an example method for generatingdroplets comprising cell beads and nucleic acid barcode molecules isschematically depicted in FIG. 16A. The method described in FIG. 16Acomprises three phases 1610, 1620, and 1630; with each respective phasecomprising: (1) generation of cell beads (1610); (2) cell bead solventexchange and processing (1620); and (3) co-partitioning of cell beadsand barcodes for subsequent tagging (e.g., barcoding) of one or moreconstituents of (or derived from) the cell bead (1630).

With continued reference to FIG. 16A, phase 1610 comprises providing anoil 1601, polymeric or gel precursors 1602, and cells or nuclei 1603(e.g., a cell, a fixed cell, a cross-linked cell, a nuclei, apermeabilized nuclei, etc.) to a microfluidic chip 1604 for dropletgeneration. The polymeric or gel precursors may be electrically chargedas described in, e.g., FIGS. 11-12. Charged species (not shown in FIG.16A), such as those described elsewhere herein, may be further providedto microfluidic chip 1604 for co-partitioning. As detailed in FIG. 16B,the microfluidic chip 1604 comprises a plurality of reservoirscomprising the oil 1601, polymeric or gel precursors 1602 and cells1603. Microfluidic chip 1604 may also comprise one or more additionalreservoirs (not shown) comprising one or more additional reagents.Polymeric or gel precursors 1602 and cells 1603 are flowed (e.g., viathe action of an applied force, such as negative pressure via a vacuumor positive pressure via a pump) from their reservoirs to a firstchannel junction and combine to form an aqueous stream. This aqueousstream is then flowed to a second channel junction, in which oil 1601 isprovided. The aqueous stream provided from the first channel junction isimmiscible with the oil 1601 resulting in the generation of a suspensionof aqueous droplets in the oil 1605, which then flow to a reservoir forcollection. Flow can be controlled within the microfluidic chip 1604 viaany suitable method, including the use of one or more flow regulators ina channel or various channels, dimensioning of microfluidic channels,etc., as described elsewhere herein. As shown in both FIG. 16A and FIG.16B, the product comprises droplets 1605 comprising a cell from thecells 1603 and polymeric or gel precursors 1602. In some cases, at leastsome of the droplets of droplets 1625 comprise a single cell.

In some cases, the droplets 1605 are subjected to conditions sufficientto lyse the cells or nuclei comprised therein, releasing cellularmacromolecular constituents into the droplets 1605. The macromolecularconstituents (e.g., nucleic acids, proteins, etc.) may be subjected toone or more reactions for additional processing. Processing ofmacromolecular constituents is described in more detail elsewhereherein. In other embodiments, the droplets 1605 are subjected toconditions sufficient to permeabilize the cells (or nuclei) therebyfacilitating access to one or more macromolecular constituents of thecell (or nucleus) for further processing. The droplets 1605 are then besubjected to conditions suitable to polymerize or gel the polymeric orgel precursors 1602 in the droplets 1605, to generate cell beads 1606.

Continuing with FIG. 16A, the droplets 1605 are then subjected toconditions suitable to polymerize or gel the polymeric or gel precursors1602 in the droplets 1605, which generates cell beads 1606 thatencapsulate the cells (or nuclei) 1603. As the resulting cell beads 1606are suspended in oil, phase 1620 is initiated which comprises a solventexchange configured to resuspend the cell beads 1606 in an aqueous phase1611. Additional details and examples regarding solvent exchange areprovided elsewhere herein.

The resuspended aqueous cell beads 1611 can then, in bulk, be optionallyprocessed 1612 to prepare the cell beads for analysis of one or morecellular components. For example, in 1612, cell beads 1611 can besubjected conditions suitable to lyse or permeabilize cells (or nuclei)in the cell beads 1613, thereby releasing or otherwise allowing accessto one or more cellular constituents (e.g., nucleic acids, such as mRNAand gDNA, proteins, etc.). Separately or contemporaneously from celllysis, cell beads (e.g., 1611 or 1613) are also subjected, in bulk, toconditions to denature nucleic acids derived from the cells (e.g., gDNA)associated with the cell beads 1611. The polymeric matrix of the cellbeads 1613 effectively hinders or prohibits diffusion of largermolecules, such as nucleic acids and/or proteins, from the cell beads1613. In addition, in cases where charged species are introduced intothe polymer matrix, the electric charge (e.g., positive charge) of thecell beads effectively prevents diffusion of molecules of oppositecharge (e.g., nucleic acid). The cell beads 1613 are sufficiently porousto facilitate diffusion of denaturation agents into the cell bead matrixto contact the nucleic acids within the cell beads 1613. In some cases,the cell beads (1611 or 1613) can then be subjected, in bulk, toconditions suitable for performing one or more reactions on nucleicacids or other analytes derived from the cells associated with the cellbeads (1611 or 1613). For example, antibodies may be washed into and/orout of the resuspended cell beads (1611 or 1613). After processing 1612,the resulting cell beads 1613 are then collected 1614 and can be storedprior to initiation of phase 1630.

In phase 1630, droplets comprising the cell beads (1611 or 1613) andbarcode beads 1622 (e.g., gel beads comprising nucleic acid barcodemolecules attached thereto) are generated. As shown in FIG. 16A, an oil1621, the cell beads 1613, and barcode beads 1622 each comprising abarcode sequence (e.g., each bead comprising a unique barcode sequence)are provided to a microfluidic chip 1623. An example microfluidic chip1623 is shown in FIG. 16C. As shown in FIG. 16C, the microfluidic chip1623 comprises a plurality of reservoirs comprising the oil 1621, cellbeads 1613 and barcode beads 1622 (e.g., gel beads). The chip alsoincludes additional reservoirs 1627 and 1628 that may be used to supplyadditional reagents (e.g., reagents for nucleic acid amplification,reagents that can degrade or dissolve cell beads and/or gel beads,reagents that degrade linkages between barcodes and gel beads, etc.).Cell beads 1613 and barcode beads 1622 are flowed (e.g., via the actionof an applied force, such as negative pressure via a vacuum or positivepressure via a pump) from their reservoirs to a first channel junctionand form an aqueous mixture. Materials from reservoirs 1627 and 1628 canalso be provided to the aqueous mixture at the first channel junction.

Alternatively, cell beads and gel beads can be mixed before introductioninto the microfluidic chip. In this case, a single reservoir of themicrofluidic chip 1623 comprises a mixture of cell beads and gel beads.The ratio of cell beads to gel beads in the mixture can be varied toalter the number of droplets generated that comprise a single cell beadand a single gel bead. The mixture of cell beads and gel beads may beflowed (e.g., via the action of an applied force, such as negativepressure via a vacuum or positive pressure via a pump) from thereservoir to a first channel junction, in some cases together withmaterials from reservoirs 1627 and/or 1628. As an alternative or inaddition to, cells may be mixed with gel beads. For example, acollection of cells and cell beads may be mixed with gel beads, or acollection of cells may be mixed with gel beads.

In some embodiments, the aqueous mixture comprising cell beads 1613,barcode beads 1621, and in some cases additional reagents is then flowedto a second channel junction, to which oil 1621 is provided. The aqueousmixture provided from the first channel junction is immiscible with theoil 1621 resulting in the generation of a suspension of aqueous droplets1625 in the oil which then flow to a reservoir for collection. Themicrofluidic chip can also include a reservoir 1629 that can acceptexcess oil from the stream emerging from the second channel. Flow can becontrolled within the microfluidic chip 1623 via any suitable strategy,including the use of one or more flow regulators (see FIGS. 16C and 16D)in a channel or that connect channels, use of various channels,dimensioning of channels, etc. As shown in both FIG. 16A and FIG. 16C,the droplets 1625 comprise a cell bead 1613 and a barcode bead 1622(e.g., a gel bead), in addition to any other reagents provided byreservoirs 1627 and 1628. In some cases, at least some droplets ofdroplets 1625 comprise a single cell bead and a single barcode bead(e.g., a single gel bead).

Where reagents that degrade or dissolve the cell beads 1613, barcodedbeads 1622 (e.g., gel beads) and/or linkages between barcodes andbarcoded beads 1622 (e.g., gel beads) are present in droplets, thesereagents can release the nucleic acids trapped in the cell beads 1613from the cell beads 1613 and/or release the barcodes from the barcodebeads 1622. The nucleic acid barcode molecules then interact with thereleased cellular components (e.g., cellular nucleic acids) to generatebarcoded nucleic acid molecules for nucleic acid sequencing as describedelsewhere herein. In embodiments where the barcode bead (e.g., gel bead)is degraded or nucleic acid barcode molecules are releasably attached tothe barcode bead (e.g., gel bead), the barcoded cellular components(e.g., barcoded cDNA or gDNA fragments) are not attached to the bead.Where a given droplet comprises a single cell bead and a single barcodedbead comprising nucleic acid barcode molecules comprising a commonbarcode sequence, the barcoded cellular components (or derivativesthereof) can be associated with the cell (or other biological sample,such as a bacterium or virus) of the given cell bead via the commonbarcode sequence.

FIG. 16D depicts two example microfluidic reactions demonstrating thegeneration of droplets 1625 comprising cell beads and gel beads usingthe method of FIG. 16A and the microfluidic devices depicted in FIGS.16B and 16C. FIG. 16D (panel A) shows droplets comprising cell beads andgel beads while FIG. 16D (panel B) shows droplets comprising cell beadscomprising magnetic materials (e.g., magnetic particles) and gel beads.

Partitions comprising a barcode bead (e.g., a gel bead) associated withbarcode molecules and a bead encapsulating cellular constituents (e.g.,a cell bead) such as cellular nucleic acids can be useful in constituentanalysis as is described in U.S. Patent Publication No. 2018/0216162,which is herein incorporated by reference in its entirety for allpurposes. Generation of a partition comprising a barcode bead and a cellbead is schematically depicted in FIG. 17. The cell bead is generated inoperation 1701 by encapsulating a cell in a polymer matrix to form thecell bead. The cell is then lysed in operation 1702 such that thenucleic acids, and other constituents of the cell, are released from thecell and entrapped by the cell bead polymer matrix. The cell bead isthen processed in conditions suitable to, e.g., digest proteins and/ordenature nucleic acids (e.g., via an alkaline reagent) in operation1703. The cell beads can then be washed and isolated for furtherprocessing.

Computer Systems

The present disclosure provides computer systems that are programmed toimplement methods of the disclosure. FIG. 18 shows a computer system1801 that is programmed or otherwise configured to (i) control amicrofluidics system during the formation of the droplets, e.g., therate of adding each component at different channels, (ii) control thereaction conditions for the click chemistry reaction inside droplets,and (iii) perform sequencing applications. The computer system 1801 canregulate various aspects of the present disclosure, such as, forexample, regulating the rate of adding various reagents, e.g., thereducing agent, and regulating the fluid flow rate in one or morechannels in a microfluidic structure when forming the droplets. Thecomputer system 1801 can be an electronic device of a user or a computersystem that is remotely located with respect to the electronic device.The electronic device can be a mobile electronic device.

The computer system 1801 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 1805, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 1801 also includes memory or memorylocation 1810 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 1815 (e.g., hard disk), communicationinterface 1820 (e.g., network adapter) for communicating with one ormore other systems, and peripheral devices 1825, such as cache, othermemory, data storage and/or electronic display adapters. The memory1810, storage unit 1815, interface 1820 and peripheral devices 1825 arein communication with the CPU 1805 through a communication bus (solidlines), such as a motherboard. The storage unit 1815 can be a datastorage unit (or data repository) for storing data. The computer system1801 can be operatively coupled to a computer network (“network”) 1830with the aid of the communication interface 1820. The network 1830 canbe the Internet, an internet and/or extranet, or an intranet and/orextranet that is in communication with the Internet. The network 1830 insome cases is a telecommunication and/or data network. The network 1830can include one or more computer servers, which can enable distributedcomputing, such as cloud computing. The network 1830, in some cases withthe aid of the computer system 1801, can implement a peer-to-peernetwork, which may enable devices coupled to the computer system 1801 tobehave as a client or a server.

The CPU 1805 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 1810. The instructionscan be directed to the CPU 1805, which can subsequently program orotherwise configure the CPU 1805 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 1805 can includefetch, decode, execute, and writeback.

The CPU 1805 can be part of a circuit, such as an integrated circuit.One or more other components of the system 1801 can be included in thecircuit. In some cases, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 1815 can store files, such as drivers, libraries andsaved programs. The storage unit 1815 can store user data, e.g., userpreferences and user programs. The computer system 1801 in some casescan include one or more additional data storage units that are externalto the computer system 1801, such as located on a remote server that isin communication with the computer system 1801 through an intranet orthe Internet.

The computer system 1801 can communicate with one or more remotecomputer systems through the network 1830. For instance, the computersystem 1801 can communicate with a remote computer system of a user(e.g., operator). Examples of remote computer systems include personalcomputers (e.g., portable PC), slate or tablet PC's (e.g., Apple® iPad,Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone,Android-enabled device, Blackberry®), or personal digital assistants.The user can access the computer system 1801 via the network 1830.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 1801, such as, for example, on thememory 1810 or electronic storage unit 1815. The machine executable ormachine readable code can be provided in the form of software. Duringuse, the code can be executed by the processor 1805. In some cases, thecode can be retrieved from the storage unit 1815 and stored on thememory 1810 for ready access by the processor 1805. In some situations,the electronic storage unit 1815 can be precluded, andmachine-executable instructions are stored on memory 1810.

The code can be pre-compiled and configured for use with a machinehaving a processor adapted to execute the code or can be compiled duringruntime. The code can be supplied in a programming language that can beselected to enable the code to execute in a pre-compiled or as-compiledfashion.

Aspects of the systems and methods provided herein, such as the computersystem 901, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such as memory (e.g., read-only memory, random-accessmemory, flash memory) or a hard disk. “Storage” type media can includeany or all of the tangible memory of the computers, processors or thelike, or associated modules thereof, such as various semiconductormemories, tape drives, disk drives and the like, which may providenon-transitory storage at any time for the software programming. All orportions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also may be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 1801 can include or be in communication with anelectronic display 1835 that comprises a user interface (UI) 1840 forproviding, for example, the extent of hydrogels formation and the swellratio of the hydrogels. Examples of UIs include, without limitation, agraphical user interface (GUI) and web-based user interface. Methods andsystems of the present disclosure can be implemented by way of one ormore algorithms. An algorithm can be implemented by way of software uponexecution by the central processing unit 1805. The algorithm can, forexample, performing sequencing, and adjusting the addition of variousreagents according to the extent of certain reactions.

Devices, systems, compositions and methods of the present disclosure maybe used for various applications, such as, for example, processing asingle analyte (e.g., RNA, DNA, or protein) or multiple analytes (e.g.,DNA and RNA, DNA and protein, RNA and protein, or RNA, DNA and protein)form a single cell. For example, a biological particle (e.g., a cell orcell bead) is partitioned in a partition (e.g., droplet), and multipleanalytes from the biological particle are processed for subsequentprocessing. The multiple analytes may be from the single cell. This mayenable, for example, simultaneous proteomic, transcriptomic and genomicanalysis of the cell.

Example 1 Syntheses of Monomers and Polymers with Click ChemistryMoieties

In some cases, a carboxylic acid group is introduced into a polymer asan anchor to attach click chemistry moieties/precursors. As shown inScheme 3, an acid-containing polymer 1C can be made by reaction ofmonomer 1A with monomer 1B in the presence of an initiator. Integers mand n are greater than 1.

When about 1 wt % of monomer 1B is used relative to monomer 1A in thepresence of about 1.6 M of NaF (about 1:1 ratio of NaF: total monomers)and a thermal initiator (e.g., 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, VA-044 from Wako, about 0.1 wt %) attemperatures of from about 30° C. to about 50° C., a polymer of numberaverage molar mass (M_(n)) of about 156K can be obtained with apolydispersity index (MW/M_(n)) of about 1.786.

According to Scheme 4A, polymer 1C can couple with propargyl amine 1D toafford polymer 1E, which can bear a plurality of click chemistrymoieties (alkynes). The coupling agent can be any coupling agent to forman amide bond from an acid and an amine. For example, EDCI, HOBt, orHATU. The reaction can be conducted under controlled pH, such as, forexample, from about pH 5.0 to about pH 9.0, from about pH 6.0 to aboutpH 8.0, from about pH 6.5 to about pH 7.5, about pH 5.0, about pH 5.5,about pH 6.0, about pH 6.5, about pH 7.0, about pH 7.5, about pH 8.0,about pH 8.5, or about pH 9.0.

Alternatively, polymer 1E can be formed from monomer 1A and monomer 1Fin a polymerization reaction shown in Scheme 5. The monomer 1F can beformed by a coupling reaction between monomer 1A and propargyl amine 1Din the presence of a coupling reagent similar to the one used in Scheme4. In some cases, about 1.5 wt % of monomer 1F relative to monomer 1A inpresence of about 0.1 wt % of AIBN and 1.2 M of NaF at 30 C can formpolymer 1E.

Syntheses of azide-containing polymers can take at least two differentroutes as well. One route, similar to Scheme 4A for propargyl-containingpolymers, is to couple the carboxylic acid of polymer 1C to a primaryamine 1DD bearing an azide functionality using an amide coupling agentto provide an azide-containing polymer 1EE, as shown in Scheme 4B.

Alternatively, the primary amine bearing an azide functionality can bean p-azido-picolyl compound 1DP which can be coupled to polymer 1C usingan amide coupling agent to provide p-azido-picolyl containing polymer1EF, as shown in Scheme 4C.

As depicted in FIG. 29 and described elsewhere herein, thep-azido-picolyl functionality is capable of chelating Cu(I)/Cu(II)thereby facilitating copper-catalyzed click-chemistry crosslinkingbetween the azide-picolyl and the corresponding alkyne linker withsignificantly lower concentrations of copper ion present. Asdemonstrated in Example 11, the use of lower copper concentrationsresults in improved biocompatibility (e.g., reduced RNA degradation)which greatly improves gene expression analysis and other biologicalassays carried in the presence of these crosslinked polymers.

Also, as described elsewhere herein, in some embodiments crosslinking ofpolymers can be carried out using copper-free click chemistry reactions.For example, the copper-free strain-promoted azide-alkyne clickchemistry reaction (SPAAC) (see, e.g., Chem. Commun., 2011, 47:6257-6259and Nature, 2015, 519(7544):486-90) can be used in which anazide-modified linker reacts with a dibenzocyclooctyne-amine(DBCO)-modified linker to form a click chemistry linkage as shown inFIG. 30. An azide-modified linker 1EE attached to polyacrylamide polymercan be used as described above in Scheme 4B. The DBCO-modified linkerattached to a polyacrylamide 1EG can be prepared using a sulfonatedDBCO-analogue 1DG as shown in Scheme 4D.

Another route is to polymerize monomer 1A with an azide-containingacrylamide. Common to both syntheses can be an amine reagent 1H as shownin Scheme 6. The amine reagent 1H can couple with polymer 1C to affordazide-containing polymer 1J. Additionally, the amine reagent 1H cancouple with acryloyl chloride to produce monomer 1I, which canpolymerize with monomer 1A to give polymer 1J containing an azide group.

A propargyl-containing monomer 1M can be synthesized as shown in Scheme7A. Propargyl alcohol can react with carbonyl-diimidazole to affordpropargylating agent 1K. Mono-propargylation of cystamine 1L withpropargylating agent 1K can provide mono-propargylated cystamine 1KL,which can be further acylated on the free amine to providedisulfide-linked, propargylated monomer 1M. Monomer 1M is a degradablealkyne-containing monomer.

Alternatively, a propargyl-containing monomer 1Q that does not include adisulfide linkage can be synthesized as shown in Scheme 7B. The monomer1Q, however, contains a carbamate linkage that is labile. As describedelsewhere herein, the monomer 1Q can be used in to form crosslinkedpolymers that do not degrade in the presence of DTT but can beselectively degraded in the presence of DETA and heat.

The synthesis of Scheme 7B is essentially the same as Scheme 7A, howeverthe reagent 1,6-dihexylamine 1R, rather than cystamine 1L, is reactedwith propargylating agent 1K to provide a mono-propargylated hexylaminecarbamate 1KR. The free amine of 1KR can then be further acylated on toprovide the carbamate-linked, propargylated monomer 1Q.

An azide-containing monomer 1N can be made from p-azidoaniline viaacylation as shown in Scheme 8.

Each of the monomers 1M, 1N can undergo polymerization with monomer 1Ato produce the corresponding polymers bearing click chemistry moieties.

Experiment 1: Preparation of copper (II) reagent in the oil phase.

1) Suspension of copper (II) salt. About 2.50 mM Krytox-COOH and about1.25 mM Cu(OAc)₂ are stirred in HFE-7500 for about 48 hours at roomtemperature.

2) Preparation of GB Oil. GB Oil can be prepared by mixing about 2.50 mMbis-Krytox-ethylene glycol-polymer (Krytox-PEG-Krytox or BKEP or FormulaI) and about 0.25 mM Krytox in solvent HFE-7500 engineered oil. GB Oilcan be pre-formulated in bulk.

3) Preparation of the oil phase solution CB Oil. The suspension ofcopper (II) salt and the GB oil can be combined together in v/v=1:1ratio, and the resulting mixture can be stirred at room temperature forabout 1 hour and filtered through a 0.22 μm PES filter to removeinsoluble copper (II) salts. The filtrate is CB Oil comprising 1.25 mMBKEP, about 1.375 mM Krytox-COOH, and about 0.625 mM copper (II) inHFE-7500. Concentration of copper (II) in aqueous layer can be analyzedusing UV-vis absorption spectroscopy (maximum absorbance wavelength atabout 286 nm).

Experiment 2: Preparation of the Aqueous Phase Solution.

A stock solution 1 of the aqueous phase solution can be prepared bymixing polymer pairs of azide-containing and alkyne-containing polymers(3.5% w/v), F-108 (0.5% w/v), magnetic particles (0.12% w/v), THPTA(0.25 mM) in water.

A stock solution 2 of the aqueous phase solution can be prepared bymixing polymer pairs of azide-containing and alkyne-containing polymers(3.5% w/v), F-108 (0.5% w/v), magnetic particles (0.12% w/v), THPTA(0.25 mM), and sodium ascorbate (156 mM) in water.

Experiment 3: Click Chemistry and Gelation in the Absence of Cells.

To make emulsions of discrete droplets, an equipment setup similar tothat depicted in FIG. 7 can be employed. Specifically, Stock solution 1(30 μL from Experiment 2) can be fed through channel 701; stock solution2 (40 μL, from Experiment 2) can be fed through channel 702; and CB Oil(200 μL, from Experiment 1) can be fed through channel 704. A collectionof water-in-oil emulsions of droplets can be obtained in a collectingwell. The emulsions of droplets can be kept in the well (with a cover)for about 60 minutes. Subsequent solvent exchange can convert the oilphase into an aqueous phase. Gelation can be observed visually and undermicroscope. Swell ratio of the gels can be measured by comparing sizedata between monodisperse in aqueous phase (100 minutes) andmonodisperse in NaOH phase (5 minutes) under microscope.

Experiment 4: Click Chemistry and Gelation in the Presence of Cells.

To make emulsions of discrete droplets, an equipment setup similar tothat depicted in FIG. 7 can be employed. Specifically, Stock solution 1(30 μL, from Experiment 2) can be fed through channel 701; stocksolution 2 (40 μL, from Experiment 2) and cells (100 cells/μL) can befed through channel 702; and CB Oil (200 μL, from Experiment 1) can befed through channel 704. A collection of water-in-oil emulsions ofdroplets can be obtained in a collecting well. The emulsions of dropletscan be kept in the well (with a cover) for about 60 minutes. Subsequentsolvent exchange can convert the oil phase into an aqueous phase (5 mMEDTA). Gels can be washed by phosphate-buffered-saline (3×). Gelationcan be observed. Single cell trapped gels can be observed undermicroscope (e.g., FIG. 19). Swell ratio of the gels can be measured bycomparing size data between monodisperse droplets in oil phase (100minutes) and monodisperse beads in aqueous phase (5 minutes).

Experiment 5: Polymerization using AIBN as Initiators

A copolymer poly(acrylamide) with click chemistry precursors attachedcan be synthesized through free radical solution polymerizations asfollows: acrylamide monomers (50 mmol, a mixture of acrylamide and aderivative thereof comprising click chemistry moieties), NaF (1.6 M) and2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044, 0.05mmol) can be dissolved in water. The mixture can be bubbled by nitrogenfor half an hour and subjected to 30° C. to 40° C. for 24 hours. Aftercooling down to room temperature, the desired product can be obtainedbased on the solubility thereof in aqueous vs. organic solvents (hexane,EtOAc, and EtOH, etc.).

Example 2 Syntheses of a Propargylated Monomer and Co-Polymer

A propargyl-containing monomer 2D was synthesized as shown in Scheme 9.Propargyl alcohol was reacted with carbonyl-diimidazole to affordpropargylating agent 2A, as shown. Mono-propargylation of cystamine 2Bwith propargylating agent 2A provided mono-propargylated cystamine 2C,which was further acylated on the free amine to providedisulfide-linked, propargylated monomer 2D. Monomer 2D is a degradablealkyne-containing monomer.

Copolymers with click chemistry precursors attached were synthesizedthrough free radical solution polymerizations as following (see Scheme10 and Scheme 11).

For a first co-polymer, acrylamide monomers (50 mmol, a mixture ofacrylamide and monomer 2D), NaF (1.6 M) and2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIBN, 0.05mmol) were dissolved in water. The mixture was bubbled with nitrogen for30 minutes, then subjected to 30° C. to 40° C. for 24 hours. Aftercooling to room temperature, the desired product was obtained based onthe solubility thereof in aqueous vs. organic solvents (hexane, EtOAc,EtOH, etc.).

For a second co-polymer, poly(acrylamide) polymers and DMTMM weredissolved in water and the pH corrected to 7.5 with NaOH.3-azido-1-propanamine was added and the solution was stirred at roomtemperature, protected from light, for about 16 hours. The product waspurified by dialysis and lyophilized to afford the target co-polymer asa white solid.

Example 3 Use of Cell Beads Generated with Click Chemistry for SingleCell DNA Sequencing

Synthesis of azide and alkyne polymers was performed as described inExample 2.

Preparation of Copper (II) Reagent in the Oil Phase

1) Suspension of copper (II) salt. About 1.375 mM Krytox-COOH and about1.25 mM Cu(OAc)₂ were stirred in HFE-7500 for about 48 hours at roomtemperature.

2) Preparation of GB Oil. GB Oil was prepared by mixing about 2.50 mMbis-Krytox-ethylene glycol-polymer (Krytox-PEG-Krytox or BKEP or FormulaI) and about 0.25 mM Krytox in solvent HFE-7500 engineered oil. GB Oilwas pre-formulated in bulk.

3) Preparation of the oil phase solution CB Oil. The suspension ofcopper (II) salt and the GB Oil were combined together in v/v=1:1 ratio,and the resulting mixture was stirred at room temperature for about 1hour and filtered through a 0.22 μm PES filter to remove insolublecopper (II) salts. The filtrate was CB Oil comprising 1.25 mM BKEP,about 1.375 mM Krytox-COOH, and about 0.625 mM copper (II) in HFE-7500.

Preparation of the aqueous phase solution

Stock solutions comprising sodium ascorbate were prepared by mixingpolymer pairs of azide-containing (1.75% w/v) and alkyne-containingpolymers (1.75% w/v), F-108 (0.5% w/v), magnetic particles (as indicatedin Table 2), THPTA (1.00 mM) in water, sodium ascorbate (10.00 mM), andoptionally DMSO (as indicated in Table 2) in water. Corresponding stocksolutions without sodium ascorbate were generated for each sample typefor use in cell bead generation (see below).

TABLE 2 Sample ST240_0.12_ ONT500_0.12_ AN400_0.06_ Name ST240_0.12 DMSOONT500_0.12 DMSO AN400_0.06 DMSO Magnetic Sphero ™ Sphero ™ Ocean OceanNanotech- AccuNano AccuNano Particles Carboxyl Carboxyl Nanotech- MonoMag Bead ™ COOH Bead ™ COOH (type) Magnetic Magnetic Mono Mag carboxylicMagnetic Magnetic Particles Particles acid beads Nanobeads NanobeadsMagnetic .12 .12 .12 .12 .06 .06 Particles (% w/v) DMSO 0 5 0 5 0 5 (%w/v)

Click Chemistry and Gelation in the Presence of Cells

To make emulsions of discrete droplets, an equipment setup similar tothat depicted in FIG. 7 can be employed. Specifically, stock solutionswith sodium ascorbate (60 μL) and BJ cells (ATCC®) were fed through afirst channel (e.g., 701); the corresponding stock solution withoutsodium ascorbate (40 μL) was fed through a second channel (e.g., 702);and Copper-free oil (270 μL) was fed through a third channel (e.g.,704). A collection of water-in-oil emulsions of droplets was obtained ina collecting well. The emulsions of droplets were kept in the well (witha cover) for about 15 minutes with shaking at 1000 rpm. Then, CB Oil wasadded to a final copper concentration of 0.9 mM. The emulsions ofdroplets were kept in the well for an additional 45 minutes with shakingat 1000 rpm. Subsequent solvent exchange was used to convert the oilphase into an aqueous phase (5 mM EDTA). Gels were washed inphosphate-buffered-saline (3×), thereby generating cell beads. Gelationwas observed visually and under microscope.

DNA Sequencing

Resultant cell beads were processed for DNA sequencing as describedelsewhere herein. Briefly, cell beads were partitioned into dropletswith barcode beads, lysed, and DNA from the cells was barcoded.Resultant barcoded DNA was isolated and subjected to nucleic acidsequencing. Nucleic acid sequencing results for each sample wereanalyzed and compared to target specifications for optimal cell beadperformance. Results of this analysis are shown in Table 3.

TABLE 3 Sample Target ST240_0.12_ ONT500_0.12_ AN400_0.06_ DescriptionSpec ST240_0.12 DMSO ONT500_0.12 DMSO AN400_0.06 DMSO Cells 131 125 224183 165 169 detected Fraction ≥0.85 0.85 0.87 0.85 0.84 0.85 0.86Observed Barcodes on Whitelist Total ≤0.40 0.71 0.83 0.65 0.69 0.51 0.46Wasted Data Fraction ≤0.10 0.16 0.22 0.12 0.16 0.09 0.08 Reads in EmptyBarcodes Amp Rate ≥0.11 0.04 0.03 0.03 0.04 0.08 0.14 Cell BarcodesMedian Amp 0.43 0.45 0.42 0.49 0.49 0.44 Rate CV DPCV Cell ≤0.13 0.110.11 0.1 0.11 0.1 0.1 Barcodes Median Unnormalized Fraction of cells≥0.97 0.98 0.97 0.99 0.98 0.97 0.97 with dpcv <0.2 Fraction of cells≥0.90 0.91 0.94 0.96 0.9 0.95 0.92 dpcv <0.15 Technical noise ≤0.15 0.050.08 0.05 0.11 0.1 0.11 fraction GC bias metric ≤0.08 0.04 0.03 0.030.03 0.04 0.03 (cells only) Diffusion Dup ≤0.10 0.08 0.09 0.08 0.08 0.070.06 Rate Full Coverage Dup Ratio 1.43 1.56 1.39 1.39 1.19 1.13 CellBarcodes Median Fraction reads 0.15 0.13 0.15 0.16 0.15 0.14non-whitelist barcodes Median 0.6 0.55 0.61 0.62 0.72 0.76 mapQ 30fraction in cells

FIG. 20 shows sequencing results from one sample, AN400_0.06_DMSO,demonstrating high quality sequencing results using these conditions.

Example 4 Cell Centering

Synthesis of azide and alkyne polymers was performed as described inExample 2.

Preparation of Copper (II) Reagent in the Oil Phase

1) Suspension of copper (II) salt. About 1.375 mM Krytox-COOH and about1.25 mM Cu(OAc)₂ were stirred in HFE-7500 for about 48 hours at roomtemperature.

2) Preparation of GB Oil. GB Oil was prepared by mixing about 2.50 mMbis-Krytox-ethylene glycol-polymer (Krytox-PEG-Krytox or BKEP or FormulaI) and about 0.25 mM Krytox in solvent HFE-7500 engineered oil. GB Oilwas pre-formulated in bulk.

3) Preparation of the oil phase solution CB Oil. The suspension ofcopper (II) salt and the GB Oil were combined together in v/v=1:1 ratio,and the resulting mixture was stirred at room temperature for about 1hour and filtered through a 0.22 μm PES filter to remove insolublecopper (II) salts. The filtrate was CB Oil comprising 1.25 mM BKEP,about 1.375 mM Krytox-COOH, and about 0.625 mM copper (II) in HFE-7500.

Preparation of the Aqueous Phase Solution

Stock solutions comprising sodium ascorbate were prepared by mixingpolymer pairs of azide-containing (1.75% w/v) and alkyne-containingpolymers (1.75% w/v), F-108 (0.5% w/v), magnetic particles (0.12% w/v),THPTA (1.00 mM) in water, and sodium ascorbate (150.00 mM) in water.Corresponding stock solutions without sodium ascorbate were generatedfor each sample type for use in cell bead generation (see below).

Click Chemistry and Gelation in the Presence of Cells

To make emulsions of discrete droplets, an equipment setup similar tothat depicted in FIG. 7 can be employed. Specifically, stock solutionswith sodium ascorbate (60 μL) and peripheral blood mononuclear cells(PBMCs) obtained from a subject were fed through a first channel (e.g.,701); the corresponding stock solution without sodium ascorbate (40 μL)was fed through a second channel (e.g., 702); and Copper-free oil (270μL) was fed through a third channel (e.g., 704). A collection ofwater-in-oil emulsions of droplets was obtained in a collecting well.The emulsions of droplets were separated into two sets of wells andprocessed via the workflow shown in FIG. 21A. One set of emulsions wascovered for about 15 minutes with shaking at 1000 rpm to facilitate cellcentering. The second set was covered for about 15 minutes with noshaking. Then, CB oil was added to a final copper concentration of 0.625mM. The emulsions of droplets were kept in the wells for an additional45 minutes with shaking at 1000 rpm. Subsequent solvent exchange wasused to convert the oil phase into an aqueous phase (5 mM EDTA). Gelswere washed in phosphate-buffered-saline (3×), thereby generating cellbeads. Gelation was observed visually and under microscope.

Nuclear Staining and Imaging

Cell beads were stained with SYBR® nuclear staining and imaged using afluorescent microscope. Imaging results are shown in FIG. 21B(uncentered) and 21C (centered). FIG. 22 shows the results of cellcentering analysis, demonstrating that the emulsions which underwent thecentering procedure (shaking) generated a greater number of cell beadswith cells near the center of the bead as compared with those that didnot undergo the centering procedure.

Example 5 Cell Bead Generation Parameters Impact DNA Degradation

Emulsions were generated comprising azide and alkyne-comprisingpolymers, λDNA, and the additional components as indicated in FIG. 23.Sodium ascorbate as provided at 150 mM. Each was generated usingcopper-free oil. λDNA concentration from each was measured byquantitative PCR (qPCR). These results indicate a reduction in λDNAconcentration, due to DNA degradation, in the presence of paramagneticparticles (PMPs) and sodium ascorbate (Na Asc). Removal of PMPs from theconditions eliminates DNA degradation.

Example 6 DMSO Addition and/or PMP Choice Impacts DNA Degradation

Emulsions were generated comprising azide and alkyne-comprisingpolymers, DNA, and the additional components as indicated in FIG. 24.Each was generated using copper-containing oil. Sodium ascorbate wasprovided at 10 mM. Two different PMPs were tested; Sphero™ CarboxylMagnetic Particles (Sphero), which comprise an iron oxide coatingsurrounding a polystyrene core, and Dynabeads magnetic beads (Dyna),which comprise an iron oxide core surrounded by an outer polymercoating, each in different concentrations of DMSO as indicated in FIG.24. These results indicate that the use of Dynabeads and addition of atleast 5% DMSO can prevent DNA degradation.

Example 7 Cell Bead Generation with CuAcAc

The use of copper (II) hexafluoroacetylacetonate (CuAcAc) as a coppersource for click chemistry-mediated cell bead generation was testedunder different parameters.

FIG. 25A shows the results of cell bead generation using varying sodiumascorbate (Na Asc) concentrations, ranging from 10 mM to 200 mM. Cellbeads were generated as described herein using the following parameters:1 mM THPTA, 1 mM CuAcAc, and 2.5 mM KmPEG oil. These results demonstratethat 100 mM of sodium ascorbate provides optimal cell bead generationunder these conditions.

FIG. 25B shows the results of cell bead generation using varyinggelation times, ranging from 0 minutes to overnight (ON). Cell beadswere generated as described herein using the following parameters: 1 mMTHPTA, 1 mM CuAcAc, 2.5 mM KmPEG oil, and 100 mM sodium ascorbate. Theseresults demonstrate that 60 minutes of gelation time is the minimumneeded to achieve optimal cell bead generation under these conditions.

FIG. 25C shows the results of cell bead generation using varying THPTAconcentrations, ranging from 0.5 mM to 8 mM. Cell beads were generatedas described herein using the following parameters: 1 mM CuAcAc, 2.5 mMKmPEG oil, and 20 mM sodium ascorbate. These results demonstrate that 5mM of THPTA provides optimal cell bead generation under theseconditions.

FIG. 25D shows the results of cell bead generation using varying sodiumascorbate (Na Asc) concentrations, ranging from 2.5 mM to 100 mM. Cellbeads were generated as described herein using the following parameters:5 mM THPTA, 1 mM CuAcAc, and 2.5 mM KmPEG oil. These results demonstratethat 50 mM of sodium ascorbate provides optimal cell bead generationunder these conditions.

FIG. 26A shows the results of cell bead generation using varying CuAcAcconcentrations, ranging from 0.3125 mM to 3.75 mM. Cell beads weregenerated as described herein using the following parameters: 5 mMTHPTA, 2.5 mM KmPEG oil, and 20 mM sodium ascorbate. These resultsdemonstrate that 1 mM of CuAcAc provides optimal cell bead generationunder these conditions.

FIG. 26B shows the results of cell bead generation using varyinggelation times, ranging from 0 minutes to overnight (ON). Cell beadswere generated as described herein using the following parameters: 5 mMTHPTA, 1 mM CuAcAc, 2.5 mM KmPEG oil, and 50 mM sodium ascorbate. Theseresults demonstrate that 15 minutes of gelation time is the minimumneeded to achieve optimal cell bead generation under these conditions.

Table 4 shows the parameters identified as optimal for cell beadgeneration using CuAcAc, requiring only 15 minutes of gelation time.

TABLE 4 Component Final concentration Azide Polymer mix 1.75% w/v AlkynePolymer mix 1.75% w/v F-108 0.50% w/v Mag. Particles (PMPs) 0.12% w/vLigand (THPTA) 5.00 mM Reducing agent (Na Asc). 50.00 mM Additive (DMSO)5% v/v

Example 8 Cell Bead Generation with Cu₂OAc

The use of copper acetate (Cu₂OAc) as a copper source for clickchemistry-mediated cell bead generation was tested under differentparameters.

FIG. 27A shows the results of cell bead generation using varying THPTAconcentrations, ranging from 0.05 mM to 8 mM. Cell beads were generatedas described herein using the following parameters: 5 mM sodiumascorbate, 0.625 mM Cu₂OAc, and 2.5 mM KmPEG oil. These resultsdemonstrate that 1 mM of THPTA provides optimal cell bead generationunder these conditions. No gelation was observed for THPA concentrationsof 0.05 mM, 5 mM, and 8 mM.

FIG. 27B shows the results of cell bead generation using varying sodiumascorbate (Na Asc) concentrations, ranging from 2.5 mM to 100 mM. Cellbeads were generated as described herein using the following parameters:1 mM THPTA, 0.625 mM Cu₂OAc, and 2.5 mM KmPEG oil. These resultsdemonstrate that 50 mM of sodium ascorbate provides optimal cell beadgeneration under these conditions.

FIG. 27C shows the results of cell bead generation using varying sodiumascorbate (Na Asc) concentrations, ranging from 2.5 mM to 100 mM. Cellbeads were generated as described herein using the following parameters:1 mM THPTA, 1 mM Cu₂OAc, and 2.5 mM KmPEG oil. These results demonstratethat 20 mM of sodium ascorbate provides optimal cell bead generationunder these conditions.

FIG. 27D shows the results of cell bead generation using varying sodiumascorbate (Na Asc) concentrations, ranging from 2.5 mM to 100 mM. Cellbeads were generated as described herein using the following parameters:1 mM THPTA, 2 mM Cu₂OAc, and 2.5 mM KmPEG oil. These results demonstratethat 20 mM of sodium ascorbate provides optimal cell bead generationunder these conditions.

FIG. 28A shows the results of cell bead generation using THPTAconcentrations, ranging from 0.025 mM to 5 mM. Cell beads were generatedas described herein using the following parameters: 2 mM Cu₂OAc, 20 mMsodium ascorbate, and 2.5 mM KmPEG oil. These results demonstrate that 1mM of THPTA provides optimal cell bead generation under theseconditions.

FIG. 28B shows the results of cell bead generation using varyinggelation times, ranging from 0 minutes to overnight (ON). Cell beadswere generated as described herein using the following parameters: 1 mMTHPTA, 2 mM Cu2OAc, 2.5 mM KmPEG oil, and 20 mM sodium ascorbate. Theseresults demonstrate that 30 minutes of gelation time is the minimumneeded to achieve optimal cell bead generation under these conditions.

Table 5 shows the parameters identified as optimal for cell beadgeneration using Cu₂OAc, requiring only 30 minutes of gelation time.

TABLE 5 Component Final concentration Azide Polymer mix 1.75% w/v AlkynePolymer mix 1.75% w/v F-108 0.50% w/v Mag. Particles (PMPs) 0.12% w/vLigand (THPTA) 1.00 mM Reducing agent (Na Asc). 20.00 mM Additive (DMSO)5% v/v

Example 9 Generation of Copper Nanoparticle/Cell Complexes

Copper nanoparticles (CuNPs) were obtained from US ResearchNanomaterials Inc. 10 mg of CuNPs were suspended in 1 mL of completecell culture medium. CuNPs were sonicated for 2 minutes, then vortexedfor 10 minutes. 10 mL of complete cell culture medium was added to theCuNP suspension and centrifuged for 5 minutes at 150 g to remove largeparticles. Supernatant was collected and centrifuged for 5 minutes at1000 g. Supernatant was discarded, and the pellet was resuspended in 1mL of complete cell culture medium. The resultant CuNP suspension wassonicated in a bath sonicator for 30 minutes.

Next, cells were resuspended in complete cell medium at 10⁷ cells permL. 1 mL of CuNP dispersion was added to the cell suspension andincubated for 15 minutes at 4° C. Then, the mixture was centrifuged for5 minutes at 50 g, 4° C., mixed gently, and centrifuged again using thesame conditions. The cell pellet was washed two times with 10 mLcomplete cell culture medium, with a centrifugation at 20 g, 4° C. andremoval of supernatant following each wash. Finally, the CuNPs/cellmixture was resuspended in 500 μL, complete medium for cell beadgeneration.

Example 10 Use of Copper Nanoparticles for Cell Bead Generation

The CuNP/cell mixture is partitioned into droplets as described herein,together with polymers for cell bead generation. Polymers in dropletscomprising a cell/CuNPs complex are cross-linked via click chemistry,using the CuNPs as a catalyst, thereby generating cell beads. Polymersin droplets which do not comprise a cell/CuNPs complex are notcross-linked A population of cell beads is generated.

Example 11 Low Copper Concentration Cell Bead Generation Using PicolylPolymers

This example illustrates how cell beads (CBs) can be generated fromemulsions of discrete droplets at low copper concentrations by usingpolymers having a copper-chelating azido-picolyl functionality. Asdepicted schematically in FIG. 29, and described elsewhere herein,linkers modified with an azido-picolyl functionality can undergo acopper-catalyzed click reaction with an alkyne-modified linker to form acrosslink comprising a 1,2,3-triazole click chemistry linkage. Further,the ability of the azido-picolyl functionality to chelate copper ioneffectively raises the copper concentration at the site of thecopper-catalyzed click reaction. This increase in effective copperconcentration at the reaction site allows for a reduction in the overallcopper concentration in the reaction without a loss in the cross-linkingefficiency. A reduction in overall copper concentration in the cell beadgeneration process significantly improves biocompatibility, e.g.,degradation of RNA components.

Two sets of CB s were generated from emulsions of discrete dropletsaccording to the methods described in Example 3. One set of CBs includedpolymers having azido-picolyl functionality whereas the other set hadpolymers having the standard azide functionality. The generation of thetwo sets of CBs was carried out by copper catalyzed click chemistry at arange of copper concentrations from 0.0625 mM to 0.625 mM. The size ofthe CBs generated under the different copper concentration conditionswere determined via microscopy.

As shown in FIG. 31A, significant differences were observed in CBsformed using the picolyl polymer and those formed using the standardnon-picolyl polymer. The picolyl polymer CBs exhibited no change inswell-ratio (SR) upon reducing Cu concentration in half (from 0.625 mMto 0.3125 mM). Moreover, the picolyl polymer CBs could be generatedusing a Cu concentration of 0.0625 mM in oil at 37 C in only 30 minutesand exhibited only a small increase in SR from 0.70 to 0.81. Incontrast, the non-picolyl polymer CBs could not be generated at a Cuconcentration of 0.0625 mM and even a decrease from 0.625 mM to 0.3125mM Cu concentration resulted in a significant increase in CB swell-ratiofrom 0.70 to 1.07.

A further experiment was carried out in which CBs were generated fromemulsion of droplets containing GM12878 cells dispersed in a polymer mixcontaining either polymers with an azido-picolyl functionality at low Cuconcentration (0.15 mM or 0.20 mM) or polymers with an azido-alkyl(i.e., non-picolyl) functionality and higher Cu concentration (0.625mM). Gelation within the droplets was carried out for 45 min at RT underthe following conditions for the droplets containing the picolyl ornon-picolyl polymers: (a) picolyl polymers: 0.15 mM Cu, 8 mM sodiumascorbate; or 0.20 mM Cu, 10 mM sodium ascorbate; (b) non-picolylpolymers: 0.625 mM Cu, 10 mM sodium ascorbate. Following gelation, theemulsion was broken, and the resulting CBs were washed twice in PBS, andthen packed by centrifugation. Equivalent volumes of the distinct setsCBs (depending on copper concentration) were added to a 3′RT mix and thestandard “Single Cell 3′ v2” protocol was carried out to generate cDNAproducts (10× Genomics, Pleasanton, Calif., USA). DTT in the 3′RT mixwas used to degrade the disulfide crosslinks of the hydrogel matrix andthereby dissolve the CBs.

As shown by the results depicted in FIG. 31B, in the CBs formed viaclick chemistry crosslinking of polymers with azido-picolylfunctionality and lower copper concentrations (0.15 mM or 0.2 mM), ˜95%of genes and ˜94% of unique molecular identifiers (UMI) were detectedrelative to cell control. In contrast, in the CBs formed usingnon-picolyl polymers and a higher copper concentration (0.625 mM), only45% of genes and only 33% of UMI were detected relative to cell control.

In summary, the ability to use substantially lower Cu concentrationsduring gelation of discrete droplets to form CBs can be carried out byincorporating polymers with the azido-picolyl functionality. The use oflower copper concentration in forming the CBs results in substantiallydecreased RNA degradation and substantially improved gene detection.

Example 12 Chemical Degradation Cell Beads with Carbamate Linkages

This example illustrates how CBs generated from emulsions of discretedroplets with labile carbamate (rather than disulfide) linkages can beselectively degraded with diethyltriamine (DETA) and heat.

Emulsions of discrete droplets are generated with polymers modified withlinkers including either azide or alkyne groups capable of undergoingCuAAC click chemistry. As illustrated by the scheme depicted in FIG. 32,the linkers with the alkyne group comprise a propargyl-carbamate moietythat undergoes copper catalyzed click chemistry crosslinking reactionwith the azide linker modified polymer to form the gel matrix. Thecrosslinks forming the gel matrix comprise a 1,2,3-triazole moiety butdo not include a disulfide linkage Thus, they are no susceptible todegradation by DTT treatment. Instead, the crosslinks can be degraded bytreatment with a polyamine (e.g., DETA) and heat (e.g., 60° C.) whichacts to cleave the carbamate group as shown in FIG. 32.

CBs were generated from an emulsion of droplets as described in Example3 except that linkers comprising a propargyl-carbamate moiety (and nodisulfide linkage) as shown in FIG. 32. The resulting CBs comprising acarbamate linkage were analyzed for their degradation characteristics inthe presence of DETA and heat. Degradation was monitored using opticalmicroscopy. 30 μL, of the carbamate CBs were exposed to 200 μL solutionof 10% DETA in PBS at 60° C. and compared to carbamate CBs exposed to acontrol PBS solution without DETA. The carbamate CBs were completelydegraded after 15 minutes in 10% DETA at 60° C.

For further comparison, gel beads (GBs) that do not contain a carbamatelinkage (10× Genomics, Inc., Pleasanton, Calif., USA) were also treatedwith 10% DETA and heat and monitored microscopically for degradation. Asshown by plot of data depicted in FIG. 33, upon treatment with 10% DETAand 60° C. heat, the CBs with carbamate containing crosslinks undergoswelling from ˜60 μm to ˜110 μm in the first 10 minutes of treatment andare completely dissolved after 15 minutes of treatment. In contrast, theGBs, which have crosslinks that do not contain a carbamate, undergo nosignificant change after 15 minutes indicating no degradation in thepresence of 10% DETA and heating to 60° C.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is:
 1. A composition comprising: (a) a biologicalparticle and/or a macromolecular constituent; and (b) two or morecrosslinked polymers, wherein the crosslinks are formed by clickchemistry.
 2. The composition of claim 1, wherein the crosslinkedpolymers are a gel.
 3. The composition of claim 1, wherein thecrosslinked polymers enclose the biological particle and/ormacromolecular constituent.
 4. The composition of claim 1, wherein thecrosslinks comprise a picolyl moiety.
 5. The composition of claim 1,wherein the click chemistry is copper-catalyzed.
 6. The composition ofclaim 5, wherein the copper-catalyzed click chemistry comprises a copperconcentration selected from about 0.3 mM or less, about 0.2 mM or less,and about 0.15 mM or less.
 7. The composition of claim 1, wherein theclick chemistry is copper-free.
 8. The compositions of claim 7, whereinthe copper-free click chemistry is selected from: (a) strain-promotedazide/dibenzocyclooctyne-amine (DBCO) click chemistry; (b) inverseelectron demand Diels-Alder (IED-DA) tetrazine/trans-cyclooctene (TCO)click chemistry; (c) inverse electron demand Diels-Alder (IED-DA)tetrazine/norbonene click chemistry; (d) Diels-Alder maleimide/furanclick-chemistry; (e) Staudinger ligation; and (f)nitrile-oxide/norbonene cycloaddition click chemistry.
 9. Thecomposition of claim 8, wherein the crosslinks comprise a 1,2,3-triazolemoiety.
 10. The composition of claim 8, wherein the crosslinks comprisea dihydropyridazine moiety.
 11. The composition of claim 1, wherein thecrosslinks comprise a labile bond.
 12. The composition of claim 11,wherein the labile bond is selected from a chemically labile bond, athermally labile bond, an enzymatically labile bond, a photo-labilebond, or a combination thereof.
 13. The composition of claim 11, whereinthe labile bond is selected from a disulfide bond, a carbamate bond, apeptide bond, or a combination thereof.
 14. The composition of claim 1,wherein the composition further comprises at least one reagent attachedby click chemistry to at least one of the polymers.
 15. The compositionof claim 14, wherein the at least one reagent is an oligonucleotide. 16.The composition of claim 15, wherein the oligonucleotide comprises apoly-T sequence.
 17. A method of forming a gel comprising: combining ina partition under click chemistry reaction conditions (i) a biologicalparticle and/or a macromolecular constituent, and (ii) two or morepolymers configured to crosslink by click chemistry.
 18. The method ofclaim 17, wherein one of the polymers configured to crosslink comprisesa picolyl moiety.
 19. The method of claim 17, wherein the clickchemistry reaction conditions comprise a copper concentration selectedfrom about 0.3 mM or less, about 0.2 mM or less, and about 0.15 mM orless.
 20. The method of claim 17, wherein the click chemistry reactionconditions are copper-free.
 21. The method of claim 20, wherein the twoor more polymers are configured to crosslink by copper-free clickchemistry selected from: (a) strain-promotedazide/dibenzocyclooctyne-amine (DBCO) click chemistry; (b) inverseelectron demand Diels-Alder (IED-DA) tetrazine/trans-cyclooctene (TCO)click chemistry; (c) inverse electron demand Diels-Alder (IED-DA)tetrazine/norbonene click chemistry; (d) Diels-Alder maleimide/furanclick-chemistry; (e) Staudinger ligation; and (f)nitrile-oxide/norbonene cycloaddition click chemistry.
 22. The method ofclaim 20, wherein the click chemistry linkage comprises a 1,2,3-triazolemoiety.
 23. The method of claim 20, wherein the click chemistry linkagecomprises a dihydropyridazine moiety.
 24. The method of claim 17,wherein the crosslinks formed comprise a labile bond.
 25. The method ofclaim 24, wherein said labile bond is a chemically labile bond, athermally labile bond, an enzymatically labile bond, or a photo-labilebond.
 26. The method of claim 24, wherein said labile bond is adisulfide bond, a carbamate bond, or a peptide bond.
 27. The method ofclaim 17, wherein said partition is a well.
 28. The method of claim 17,wherein said partition is a discrete droplet in an emulsion.